Measurements of transmembrane potential and magnetic field at the apex of the heart

We studied the transmembrane potential and magnetic fields from electrical activity at the apex of the isolated rabbit heart experimentally using optical mapping and superconducting quantum interference device microscopy, and theoretically using monodomain and bidomain models. The cardiac apex has a complex spiral fiber architecture that plays an important role in the development and propagation of action currents during stimulation at the apex. This spiral fiber orientation contains both radial electric currents that contribute to the electrocardiogram and electrically silent circular currents that cannot be detected by the electrocardiogram but are detectable by their magnetic field, B_z. In our experiments, the transmembrane potential, V_m, was first measured optically and then B_z was measured with a superconducting quantum interference device microscope. Based on a simple model of the spiral structure of the apex, V_m was expected to exhibit circular wave front patterns and B_z to reflect the circular component of the action currents. Although the circular V_m wave fronts were detected, the B_z maps were not as simple as expected. However, we observed a pattern consistent with a tilted axis for the apex spiral fiber geometry. We were able to simulate similar patterns in both a monodomain model of a tilted stack of rings of dipole current and a bidomain model of a tilted stack of spiraled cardiac tissue that was stimulated at the apex. The fact that the spatial pattern of the magnetic data was more complex than the simple circles observed for V_m suggests that the magnetic data contain information that cannot be found electrically.     

Dynamic origin of spatially discordant alternans in cardiac tissue

Alternans, a condition in which there is a beat-to-beat alternation in the electromechanical response of a periodically stimulated cardiac cell, has been linked to the genesis of life-threatening ventricular arrhythmias. Optical mapping of membrane voltage (V_m) and intracellular calcium (Ca_i) on the surface of animal hearts reveals complex spatial patterns of alternans. In particular, spatially discordant alternans has been observed in which regions with a large-small-large action potential duration (APD) alternate out-of-phase adjacent to regions of small-large-small APD. However, the underlying mechanisms that lead to the initiation of discordant alternans and govern its spatiotemporal properties are not well understood. Using mathematical modeling, we show that dynamic changes in the spatial distribution of discordant alternans can be used to pinpoint the underlying mechanisms. Optical mapping of V_m and Ca_i in paced rabbit hearts revealed that spatially discordant alternans induced by rapid pacing exhibits properties consistent with a purely dynamical mechanism as shown in theoretical studies. Our results support the viewpoint that spatially discordant alternans in the heart can be formed via a dynamical pattern formation process which does not require tissue heterogeneity.     

Bench and mathematical modeling of the effects of breathing a helium/oxygen mixture on expiratory time constants in the presence of heterogeneous airway obstructions

Background

Vectorcardiogram (VCG) signals monitor both spatial and temporal cardiac electrical activities along three orthogonal planes of the body. However, the absence of spatiotemporal resolution in conventional VCG representations is a major impediment for medical interpretation and clinical usage of VCG. This is especially so because time-domain features of 12-lead ECG, instead of both spatial and temporal characteristics of VCG, are widely used for the automatic assessment of cardiac pathological patterns.

Materials and methods

We present a novel representation approach that captures critical spatiotemporal heart dynamics by displaying the real time motion of VCG cardiac vectors in a 3D space. Such a dynamic display can also be realized with only one lead ECG signal (e.g., ambulatory ECG) through an alternative lag-reconstructed ECG representation from nonlinear dynamics principles. Furthermore, the trajectories are color coded with additional dynamical properties of space-time VCG signals, e.g., the curvature, speed, octant and phase angles to enhance the information visibility.

Results

In this investigation, spatiotemporal VCG signal representation is used to characterize various spatiotemporal pathological patterns for healthy control (HC), myocardial infarction (MI), atrial fibrillation (AF) and bundle branch block (BBB). The proposed color coding scheme revealed that the spatial locations of the peak of T waves are in the Octant 6 for the majority (i.e., 74 out of 80) of healthy recordings in the PhysioNet PTB database. In contrast, the peak of T waves from 31.79% (117/368) of MI subjects are found to remain in Octant 6 and the rest (68.21%) spread over all other octants. The spatiotemporal VCG signal representation is shown to capture the same important heart characteristics as the 12-lead ECG plots and more.

Conclusions

Spatiotemporal VCG signal representation is shown to facilitate the characterization of space-time cardiac pathological patterns and enhance the automatic assessment of cardiovascular diseases.     

Transmembrane Current Imaging in the Heart during Pacing and Fibrillation

Recently, we described a method to quantify the time course of total transmembrane current (I_m) and the relative role of its two components, a capacitive current (I_c) and a resistive current (I_ion), corresponding to the cardiac action potential during stable propagation. That approach involved recording high-fidelity (200 kHz) transmembrane potential (V_m) signals with glass microelectrodes at one site using a spatiotemporal coordinate transformation via measured conduction velocity. Here we extend our method to compute these transmembrane currents during stable and unstable propagation from fluorescence signals of V_m at thousands of sites (3 kHz), thereby introducing transmembrane current imaging. In contrast to commonly used linear Laplacians of extracellular potential (V_e) to compute I_m, we utilized nonlinear image processing to compute the required second spatial derivatives of V_m. We quantified the dynamic spatial patterns of current density of I_m and I_ion for both depolarization and repolarization during pacing (including nonplanar patterns) by calibrating data with the microelectrode signals. Compared to planar propagation, we found that the magnitude of I_ion was significantly reduced at sites of wave collision during depolarization but not repolarization. Finally, we present uncalibrated dynamic patterns of I_m during ventricular fibrillation and show that I_m at singularity sites was monophasic and positive with a significant nonzero charge (I_m integrated over 10 ms) in contrast with nonsingularity sites. Our approach should greatly enhance the understanding of the relative roles of functional (e.g., rate-dependent membrane dynamics and propagation patterns) and static spatial heterogeneities (e.g., spatial differences in tissue resistance) via recordings during normal and compromised propagation, including arrhythmias.     

Transmembrane Current Imaging in the Heart during Pacing and Fibrillation

Recently, we described a method to quantify the time course of total transmembrane current (I_m) and the relative role of its two components, a capacitive current (I_c) and a resistive current (I_ion), corresponding to the cardiac action potential during stable propagation. That approach involved recording high-fidelity (200 kHz) transmembrane potential (V_m) signals with glass microelectrodes at one site using a spatiotemporal coordinate transformation via measured conduction velocity. Here we extend our method to compute these transmembrane currents during stable and unstable propagation from fluorescence signals of V_m at thousands of sites (3 kHz), thereby introducing transmembrane current imaging. In contrast to commonly used linear Laplacians of extracellular potential (V_e) to compute I_m, we utilized nonlinear image processing to compute the required second spatial derivatives of V_m. We quantified the dynamic spatial patterns of current density of I_m and I_ion for both depolarization and repolarization during pacing (including nonplanar patterns) by calibrating data with the microelectrode signals. Compared to planar propagation, we found that the magnitude of I_ion was significantly reduced at sites of wave collision during depolarization but not repolarization. Finally, we present uncalibrated dynamic patterns of I_m during ventricular fibrillation and show that I_m at singularity sites was monophasic and positive with a significant nonzero charge (I_m integrated over 10 ms) in contrast with nonsingularity sites. Our approach should greatly enhance the understanding of the relative roles of functional (e.g., rate-dependent membrane dynamics and propagation patterns) and static spatial heterogeneities (e.g., spatial differences in tissue resistance) via recordings during normal and compromised propagation, including arrhythmias.     

Calculation of optical signal using three-dimensional bidomain/diffusion model reveals distortion of the transmembrane potential

Optical mapping experiments allow investigators to view the effects of electrical currents on the transmembrane potential, V_m, as a shock is applied to the heart. One important consideration is whether the optical signal accurately represents V_m. We have combined the bidomain equations along with the photon diffusion equation to study the excitation and emission of photons during optical mapping of cardiac tissue. Our results show that this bidomain/diffusion model predicts an optical signal that is much smaller than V_m near a stimulating electrode, a result consistent with experimental observations. Yet, this model, which incorporates the effect of lateral averaging, also reveals an optical signal that overestimates V_m at distances >1 mm away from the electrode. Although V_m falls off with distance r from the electrode as exp(−r/λ)/r, the optical signal decays as a simple exponential, exp(−r/λ). Moreover, regions of hyperpolarization adjacent to a cathode are emphasized in the optical signal compared to the region of depolarization under the cathode. Imaging methods utilizing optical mapping techniques will need to account for these distortions to accurately reconstruct V_m.     

Cat Heart Muscle in Vitro : II. The steady state resting potential in quiescent papillary muscles

The steady state transmembrane resting potential difference (V_m) has been measured in quiescent papillary muscles. V_m was determined as a function of the external K concentration in Cl and SO₄ solutions and compared with the K equilibrium potential. Other measurements were made after replacement of external Na by choline, K by Rb and Cs, and Cl by SO₄, CH₃SO₄, and NO₃. Effects on V_m of albumin, temperature, and variation in internal K concentration are described.     

Leucine aminopeptidase (bovine lens): Synthesis and kinetic properties of ortho-, meta-, and para-substituted leucyl-anilides

The synthesis of a number of leucyl derivatives of substituted anilides and their properties as substrates and inhibitors of Zn²⁺-Mg²⁺ leucine aminopeptidase (EC 3.4.11.1) at pH 8.5 and 30 °C are described. The compounds include leucyl-X where X is o-, m-, or p-aminobenzenesulfonic acid, o-, m-, or p-anisidine, and m- or p-aminobenzenesulfonyl fluoride. The latter two sulfonyl fluorides, designed to be active site-directed irreversible inhibitors, turned out to be good substrates for leucine aminopeptidase. The K_m and V values of the above compounds as substrates for leucine aminopeptidase are reported. N-Leucyl-m-aminobenzenesulfonate exhibits desirable properties (solubility much greater than K_m, Δ? at 295 nm of 2000 m^?1 cm^?1, and V of 300 μmol min^?1 mg^?1) as a substrate for a spectrophotometric assay of leucine aminopeptidase. With the exception of N-leucyl-p-aminobenzenesulfonate, all of the above compounds are inhibitors of the hydrolysis of leucyl-p-nitroanilide by leucine aminopeptidase with K_i values approximately their K_m values when they are used as substrates. Despite wide variability in steric bulk, chemical composition, and electrical charge of the substituted anilides, the K_m values of the above compounds vary over a narrow range (0.5 to 4.8 mm), which indicates that the leucyl moiety plays the predominant role in the determination of K_m values. Although the K_m values of m- substituents are similar to those of o- substituents, the V values for m-substituents are much greater than those for o- substituents, which suggests that o-substituents interfere with the catalytic process. N-Leucyl-p-aminobenzenesulfonate and N-alanyl-p-aminobenzenesulfonate as well as the nonsubstrate p-aminobenzenesulfonate stimulate rather than inhibit the proteolysis of leucyl-p-nitroanilide. The stimulation has no effect on V but lowers the K_m for the hydrolysis of leucyl-p-nitroanilide, which is compatible with these compounds' serving as nonessential activators.     

Level of Thyroid-Stimulating Hormone (TSH) in Patients with Acute Schizophrenia,Unipolar Depression or Bipolar Disorder

The aim of this study is to investigate differences in thyroid-stimulating hormone (TSH) level in patients with acute schizophrenia, unipolar depression, bipolar depression and bipolar mania. Serum level of TSH was measured in 1,685 Caucasian patients (1,064 women, 63.1 %; mean age 46.4). Mean serum TSH concentration was: schizophrenia (n = 769) 1.71 μIU/mL, unipolar depression (n = 651) 1.63 μIU/mL, bipolar disorder (n = 264) 1.86 μIU/mL, bipolar depression (n = 203) 2.00 μIU/mL, bipolar mania (n = 61) 1.38 μIU/mL (H = 11.58, p = 0.009). Depending on the normal range used, the overall rate of being above or below the normal range was 7.9–22.3 % for schizophrenia, 13.9–26.0 % for unipolar depression, 10.8–27.6 % for bipolar disorder, 12.2–28.5 % for bipolar depression, and 11.4–24.5 % for bipolar mania. We have also found differences in TSH levels between the age groups (≤20, >20 years and ≤40, >40 years and ≤60 years and >60 years). TSH level was negatively correlated with age (r = ? 0.23, p < 0.001). Weak correlations with age have been found in the schizophrenia (r = ? 0.21, p < 0.001), unipolar depression (r = ? 0.23, p < 0.001), bipolar depression (r = ? 0.25, p = 0.002) and bipolar disorder (r = ? 0.21, p = 0.005) groups. Our results confirm that there may be a higher prevalence of thyroid dysfunctions in patients with mood disorders (both unipolar and bipolar) and that these two diagnostic groups differ in terms of direction and frequency of thyroid dysfunctions.     

Rye germ acid phosphatase: properties of the enzyme and its activation by lectins

Acid phosphatase (EC 3.1.3.2) from rye germs is a glycoprotein of M, 90000 with subunit structure. The pH optimum for pNPP hydrolysis is 5.4. The best substrates for the enzyme are pNPP, PP_i and ATP. In the presence of plant lectins an increase in AcPase activity was found. ConA causes a 20% decrease of K_m^app and a 50% increase of V_max^app with pNPP as substrate.     

Characterization of Neutral Lipase BT-1 Isolated from the Labial Gland of Bombus terrestris Males

Background

In addition to their general role in the hydrolysis of storage lipids, bumblebee lipases can participate in the biosynthesis of fatty acids that serve as precursors of pheromones used for sexual communication.

Results

We studied the temporal dynamics of lipolytic activity in crude extracts from the cephalic part of Bombus terrestris labial glands. Extracts from 3-day-old males displayed the highest lipolytic activity. The highest lipase gene expression level was observed in freshly emerged bumblebees, and both gene expression and lipase activity were lower in bumblebees older than 3 days. Lipase was purified from labial glands, further characterized and named as BT-1. The B. terrestris orthologue shares 88% sequence identity with B. impatiens lipase HA. The molecular weight of B. terrestris lipase BT-1 was approximately 30 kDa, the pH optimum was 8.3, and the temperature optimum was 50°C. Lipase BT-1 showed a notable preference for C8-C10 p-nitrophenyl esters, with the highest activity toward p-nitrophenyl caprylate (C8). The Michaelis constant (K_m) and maximum reaction rate (V_max) for p-nitrophenyl laurate hydrolysis were K_m = 0.0011 mM and V_max = 0.15 U/mg.

Conclusion

This is the first report describing neutral lipase from the labial gland of B. terrestris. Our findings help increase understanding of its possible function in the labial gland.     

Spatial Characterization of Electrogram Morphology from Transmural Recordings in the Intact Normal Heart

Purpose

Unipolar (UE) and bipolar electrograms (BE) are utilized to identify arrhythmogenic substrate. We quantified the effect of increasing distance from the source of propagation on local electrogram amplitude; and determined if transmural electrophysiological gradients exist with respect to propagation and stimulation depth.

Methods

Mapping was performed on 5 sheep. Deployment of >50 quadripolar transmural needles in the LV were located in Cartesian space using Ensite. Contact electrograms from all needles were recorded during multisite bipolar pacing from epicardial then endocardial electrodes. Analysis was performed to determine stimulus distance to local activation time, peak negative amplitude (V_-P), and peak-peak amplitude (V_P-P) for (1) unfiltered UE, and (2) unfiltered and 30 Hz high-pass filtered BEs. Each sheep was analysed using repeated ANOVA.

Results

Increasing distance from the pacing sites led to significant (p<0.01) attenuation of UEs (V_-P = 7.0±0.5%; V_P-P = 5.4±0.3% per cm). Attenuation of BE with distance was insignificant (V_p-p unfiltered  = 2.2±0.5%; filtered  = 1.7±1.4% per cm). Independent of pacing depth, significant (p<0.01) transmural electrophysiological gradients were observed, with highest amplitude occurring at epicardial layers for UE and endocardial layers for BE. Furthermore, during pacing, propagation was earlier at the epicardium than endocardial layer by 1.6±2.0 ms (UE) and 1.4±2.8 ms (BE) (all p>0.01) during endocardial stimulation, and 2.3±2.4 ms (UE) and 1.8±3.7 ms (BE) during epicardal stimulation (all p<0.01).

Conclusions

Electrogram amplitude is inversely proportional to propagation distance for unipolar modalities only, which affected V_-P>V_P-P. Conduction propagates preferentially via the epicardium during stimulation and is believed to contribute to a transmural amplitude gradient.     

Purification and some properties of two polyphenol oxidases from bartlett pears

Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with K_m values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. V_max and K_m values for oxygen and chlorogenic acid were 103 μmoles O₂ uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. K_m values for chlorogenic acid were independent of pH from 3 to 7; the V_max values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with K_i values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with K_i values of 0.04 and 0.11 mm for enzymes A and B, respectively.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

Purification and characterization of an intracellular N-terminal exopeptidase from Streptococcus durans

An intracellular N-terminal exopeptidase isolated from cell extracts of Streptococcus durans has been purified 470-fold to homogeneity (specific activity of 12.0 μmol/min per mg). In the absence of thiol compounds, the purified aminopeptidase undergoes a slow oxidation with a 70% loss of activity, which can be restored by the addition of 2 mM β-mercaptoethanol. The purified aminopeptidase (M_r 300 000) preferred L-peptide and arylamide substrates with small nonpolar or basic side chains. SDS electrophoresis yielded a single protein band corresponding to a molecular weight of 49 400, suggesting that the native enzyme is a hexameric protein. The enzyme-catalyzed hydrolysis of $\text{L}\text{-}\text{alanyl}\text{-p-}\text{nitroanilide}$ exhibited a bell-shaped pH dependence for log V_max/K_m(pK₁ = 6.35; pK₂ = 8.50) while the log V_max versus pH profile showed only an acid limb (pK = 6.35). Methylene blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino acid analysis indicated that this photooxidative loss of activity corresponded to the modification of one histidine residue per enzyme monomer. N-Ethylmaleimide (100 mM) caused a 78% reduction in enzyme activity. Treatment of the enzyme with 1.0 mM hydrogen peroxide resulted in the oxidation of two cysteine residues per enzyme monomer and caused a 70% decrease in the catalytic activity.     

On the amino acids involved in the ATPase site of mitochondrial F1 and the implication of the subunit C.

The data on the pH dependence of the K_m for Mg-ATP and the V_m of the ATPase of pig heart mitochondrial F₁ indicate the presence of two groups of different pK's which modify the enzyme activity. The first pK at pH 9.6 ± 0.2 may be related to the possible presence of arginine and/or tyrosine residues in the ATPase site; the second pK at pH 7.2 ± 0.2 could be due to the presence of a histidine residue in the ATPase site or to the involvement of amino groups in the ATPase site. The inhibition induced by photooxidation in the presence of Rose Bengal is not pH dependent in the pH range corresponding to the pK of histidine. The inhibition induced by diethylpyrocarbonate cannot be reversed by hydroxylamine and the characteristics of this inhibition rather correspond to the reaction of the inhibitor with amino groups. Pyridoxal phosphate also inhibits the ATPase activity of F₁ by reaction with amino groups. The presence of ATP or phosphate partially protects against the inhibition induced by diethylpyrocarbonate or pyridoxal phosphate, which indicates that amino groups may be directly or indirectly involved in the binding of nucleotide and phosphate to F₁. Glutaraldehyde also inhibits the enzyme by reacting with amino groups and inducing a crosslinking of the subunits. The disappearance of subunit C is well correlated with the decrease of ATPase activity, indicating that subunit C is essential in the ATPase activity.     

Oxalacetate decar☐ylase and pyruvate car☐ylase activities,and effect of sulfhydryl reagents in malic enzyme from Sulfolubus solfataricus

Malic enzyme (S)-malate: NADP⁺ oxidoreductase (oxaloacetate-decar☐ylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decar☐ylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decar☐ylation of l-malate. The oxaloacetate decar☐ylase activity was stimulated about 50% by NADP but only in the presence of MgCl₂, and was strongly inhibited by l-malate and NADPH which abolished the NADP activation. In the presence of MnCl₂ and in the absence of NADP, the Michaelis constant and V_m for oxaloacetate were 1.7 mM and 2.3 μmol·min⁻¹·mg⁻¹, respectively. When MgCl₂ replaced MnCl₂, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the K_m and V_m values for l-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5′-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decar☐ylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both l-malate and MnCl₂, and strongly enhanced by the car☐ylic acid substrates; NADP + malate + MnCl₂ afforded total protection. The inactivation of the oxaloacetate decar☐ylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decar☐ylase activity.     

A convenient analysis of Michaelis enzyme kinetic progress curves based on second derivatives

A simple method is described for the estimation of the Michaelis parameters, K_m and V_m, from a single progress curve at a single substrate concentration without the need to follow the reaction to completion. By measuring the substrate concentration and the time when the second derivative is at a minimum, K_m and V_m can be easily obtained.     

Reverberation of excitation in neuronal networks interconnected through voltage-gated gap junction channels

We combined Hodgkin–Huxley equations and gating models of gap junction (GJ) channels to simulate the spread of excitation in two-dimensional networks composed of neurons interconnected by voltage-gated GJs. Each GJ channel contains two fast and slow gates, each exhibiting current–voltage (I-V) rectification and gating properties that depend on transjunctional voltage (V_j). The data obtained show how junctional conductance (g_j), which is necessary for synchronization of the neuronal network, depends on its size and the intrinsic firing rate of neurons. A phase shift between action potentials (APs) of neighboring neurons creates bipolar, short-lasting V_j spikes of approximately ±100 mV that induce V_j gating, leading to a small decay of g_j, which can accumulate into larger decays during bursting activity of neurons. We show that I-V rectification of GJs in local regions of the two-dimensional network of neurons can lead to unidirectional AP transfer and consequently to reverberation of excitation. This reverberation can be initiated by a single electrical pulse and terminated by a low-amplitude pulse applied in a specific window of reverberation cycle. Thus, the model accounts for the influence of dynamically modulatable electrical synapses in shaping the function of a neuronal network and the formation of reverberation, which, as proposed earlier, may be important for the development of short-term memory and its consolidation into long-term memory.     

A convenient test for lipase activity in aqueous-based solutions

We proposed a convenient and accurate method for the measurement of lipase activity in a uniform aqueous-based substrate solution. In this work, lipase from Candida rugosa was used as the model lipase to test its catalytic ability toward p-nitrophenyl palmitate (p-NPP), which was suspended in a mixture of p-NPP ethanol solution and buffer. An ultraviolet–visible spectrophotometer was used to efficiently measure the liberated p-nitrophenol without extraction or centrifugation. Several factors that affected lipase activity were investigated, such as the ratio of p-NPP ethanol solution to buffer, the concentrations of p-NPP and lipase, as well as the temperature, reaction time, pH and agitation rate. Additionally, enzyme catalytic parameters such as K_m, V_m and “activation energy” were also assessed. We determined the optimal conditions for lipase in this homogeneous system and demonstrated lipase's catalytic performance in this condition followed Michealis–Menten kinetics.