Studies of insufficient mixing in bioreactors: Effects of limiting oxygen concentrations and short term oxygen starvation on Penicillium chrysogenum

The effect of oxygen limitation on the respiration rate of Penicillium chrysogenum was studied. The results show that measurements of critical oxygen tensions within a process that on morphological or on physical grounds exhibits an inhomogenous structure are not likely to resemble the Monod model.In order to study the effects of short term oxygen starvation on the respiratory capacity of Penicillium chrysogenum, a two compartment fermenter was constructed. This fermenter consists of one well mixed aerobic part (CSTR) and one minor anaerobic part (CPFR). In the latter the circulation time as well as the volume can be varied. After passage of the whole cell culture volume through the anaerobic part, irreversible inhibition of the respiration was observed. This was caused by a circulation time of 5 and 10 min in the plug flow reactor and with a volume of 6% of the stirred tank reactor volume. However, circulation times of 1 and 2 min with an anaerobic zone of 1% of the stirred tank reactor volume did not give any irreversible effects on the respiratory capacity.This was compared with the results of the previously established model ln(1 — I _OUR//100)^–1 = kt [1]. The I _OUR is the percentage irreversible inhibition of the respiration, t is the anaerobic circulation time and k is a constant. The two compartment fermenter results agree with the earlier model at circulation times of 5 and 10 min, but not with the shorter times, and this suggests that a lag phase exists in the inactivation kinetics.     

Interactions of cell morphology and transport processes in the lovastatin fermentation

The cholesterol lowering drug, Lovastatin (Mevacor), acts as an inhibitor of HMGCoA reductase, and is produced from an Aspergillus terreus fermentation.Pilot scale studies were carried out in 800 liter fermenters to determine the effects of cell morphology on the oxygen transport properties of this fermentation. Specifically, parallel fermentations giving (i) filamentous mycelial cells, and (ii) discrete mycelial pellets, were quantitatively characterized in terms of broth viscosity, availability of dissolved oxygen, oxygen uptake rates and the oxygen transfer coefficient under identical operating conditions.The growth phase of the fermentation, was operated using a cascade control strategy which automatically changed the agitation speed with the goal of maintaining dissolved oxygen at 50% saturation. Subsequently stepwise changes were made in agitation speed and aeration rate to evaluate the response of the mass transfer parameters (DO, OUR, and k _L a). The results of these experiments indicate considerable potential advantages to the pellet morphology from the standpoint of oxygen transport processes.List of Symbols DO % sat. Dissolved oxygen concentration - k _L a h^–1 Gas-liquid mass transfer coefficient - OUR mmol/dm³h Oxygen uptake rate - P/V KW/m³ Agitator power per unit volume - V _s m/s Superficial air velocity - _app cP Apparent viscosity     

On-line biomass estimation using a modified oxygen utilization rate

A procedure for estimating biomass during batch fermentation from on-line gas analysis is presented. First, the respiratory quotient was used to determine the fraction of the total oxygen utilization rate required for cell maintenance and growth versus product synthesis. The modified oxygen utilization rate was then used to estimate biomass on-line by integrating the oxygen balance for cell synthesis-maintenance. The method is illustrated for the case of L-lysine synthesis by Corynebacterium glutamicum.List of Symbols CER mmol CO₂/l · h carbon dioxide evolution rate - M _{O 2}/x mmol O₂/h · g cells maintenance coefficient - OUR mmol O₂/l · h oxygen utilization rate - OUR _X mmol O₂/l · h OUR fraction for cell maintenance and growth - RQ mmol CO₂/mmol O₂ respiratory quotient(CER/OUR) - X g cells/l biomass concentration - Y _X/O₂ yield coefficients     

Modifications of current properties by expression of a foreign potassium channel gene in Xenopus embryonic cells

The pH-sensitivity of transepithelial K⁺ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pH _iwas measured microfluorometrically with the pH-sensitive dye 2,7-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (I _sc), which is a measure for transepithelial K⁺ secretion, was calculated from measurements of the transepithelial voltage (V _t)and the transepithelial resistance (R _t) in a micro-Ussing chamber. All experiments were conducted in virtually HCO ₃ ^– -free solutions. Under control conditions, pH _iwas 7.01±0.04 (n=18), V _twas 9.1±0.5 mV, R _t16.7±0.09 cm², and I _sc was 587±30 A/cm² (n=49). Addition of 20 mm propionate^– caused a biphasic effect involving an initial acidification of pH _i, increase in V _tand I _sc and decrease in R _tand a subsequent alkalinization of pH _i, decrease of V _tand increase of R _t. Removal of propionate^– caused a transient effect involving an alkalinization of pH _i, a decrease of V _tand I _sc and an increase in R _t. pH _iin the presence of propionate^– exceeded pH _iunder control conditions. Effects of propionate ^– on V _t, R _tand I _sc were significantly larger when propionate^– was applied to the basolateral side rather than to the apical side of the epithelium. The pH _i-sensitivityof I _sc between pH 6.8 and 7.5 was –1089 A/(cm² · pH-unit) suggesting that K⁺ secretion ceases at about pH _i7.6. Acidification of the extracellular pH (pH _o)caused an increase of V _tand I _sc and a decrease of R _tmost likely due to acidification of pH _i. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pH _osensitivity of I _sc between pH 7.4 and 6.4 was –155 A/(cm² · pH unit). These results demonstrate that transepithelial K⁺ transport is sensitive to pH _iand pH _oand that vestibular dark cells contain propionate^– uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K⁺ channel (I _sK)in the apical membrane. Whether the effect of pH _ion the I _sK channel is a direct or indirect effect remains to be determined.The authors wish to thank Drs. Daniel C. Marcus, Zhjiun Shen and Hiroshi Sunose for helpful discussions. This work was supported by grants NIH-R29-DC01098 and NIH-R01-DC00212.     

Apical Cl− Channels in A6 Cells

Short-circuit current (I _sc ), transepithelial conductance (G _t ), electrical capacitance (C _T ) and the fluctuation in I _sc were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na⁺- and Cl⁻-free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases in G _t , C _T , I _sc and generated a Lorentzian I _sc -noise. The responses could be related to active, electrogenic secretion of Cl⁻. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable cAMP analogue or forskolin showed stable increases in G _t with only moderate peaking of I _sc . Phosphodiesterase inhibitors also stimulated Cl⁻ secretion with peaking responses in G _t and I _sc . All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl⁻ channel, with almost identical corner frequency (40–50 Hz). Repetitive challenge with the hormones led to a refractory behavior of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused C _T , a measure of apical membrane area, to increase in a manner roughly synchronous with G _t . These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular Cl⁻ channel pool. Apical flufenamate depressed Cl⁻ current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel inhibitor. Received: 20 May 1998/Revised: 8 September 1998     

Irradiance stress responses of gas exchange and antioxidant enzyme contents in pariparoba [<Emphasis Type="Italic">Pothomorphe umbellata</Emphasis> (L.) Miq.] plants

We evaluated the growth and development of the medicinal species Pothomorphe umbellata (L.) Miq. under different shade levels (full sun and 30, 50, and 70 % shade, marked as I₁₀₀, I₇₀, I₅₀, and I₃₀, respectively) and their effects on gas exchange and activities of antioxidant enzymes. Photosynthetically active radiation varied from 1 254 μmol m⁻² s⁻¹ at I₁₀₀ to 285 μmol m⁻² s⁻¹ at I₃₀. Stomatal conductance, net photosynthetic rate, and relative chlorophyll (Chl) content were maximal in I₇₀ plants. Plants grown under I₁₀₀ produced leaves with lower Chl content and signs of chlorosis and necrosis. These symptoms indicated Chl degradation induced by the generation of reactive oxygen species. Stress related antioxidant enzyme activities (Mn-SOD, Fe-SOD, and Cu/Zn-SOD) were highest in I₁₀₀ plants, whereas catalase activity was the lowest. Hence P. umbellata is a shade species (sciophyte), a feature that should be considered in reforestation programs or in field plantings for production of medicinal constituents.     

Lysosomal involvement in apoptosis

Abstract

The involvement of reactive oxygen species (ROS) in the induction of DNA damage to Escherichia coli cells caused by UVC (254 nm) irradiation was studied. We verified the expression of the soxS gene induced by UVC (254 nm) and its inhibition by sodium azide, a singlet oxygen (¹O₂) scavenger. Additional results showed that a water-soluble carotenoid (norbixin) protects against the lethal effects of UVC. These results suggest that UVC radiation can also cause ROS-mediated lethality.     

Effect of light on Synechocystis sp. and modelling of its growth rate as a response to average irradiance

The growth rate and CO₂ biofixation rate of a photosynthetic organism depend basically on the availability of light, all other factors being optimum. In dense cultures of cyanobacteria or micro-algae intended for biomass production, incident irradiance on the reactor surface is not the same as the intensity which is received by cells, as irradiance is attenuated by cell absorption and the self-shading effect. In a well-mixed, dense culture, only the average irradiance, I _av, can be considered responsible for the photosynthetic response. In this study, the photosynthetic response of Synechocystis sp., estimated from its specific growth rate, was measured for each I _av in batch cultures irradiated with different levels of external irradiance, I _ext. The specific growth rate of Synechocystis sp. depends on I _av, in accordance with the model proposed by Muller-Feuga (J Exp Mar Biol Ecol 236:1–13, 1999). A non-linear regression analysis estimated a maximum specific growth rate of 0.108 h⁻¹, at an I _av of 930 μmol photons·m⁻²·s⁻¹. This reveals that Synechocystis sp. is a highly light-tolerant strain, suitable for outdoor cultures. Higher I _av levels caused photoinhibition in batch cultures. Parameters obtained from the Muller-Feuga model show that the minimum irradiance needed to start growth mechanisms becomes less as light availability decreases, i.e. cells become more efficient in the use of light when it is scarce. This observation suggests that choosing for low-light adaptation may be a good strategy to improve productivity in dense cultures, where light is a limiting factor.     

III. Ion Channel Expression in PMA-differentiated Human THP-1 Macrophages

Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K⁺ currents, I _DR, present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K⁺ channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K⁺ channel (I _IR) and a Ca²⁺-activated maxi-K channel (I _BK). I _IR was a classical inward rectifier, conducting large inward currents negative to E _K and very small outward currents. I _IR was blocked in a voltage-dependent manner by Cs⁺, Na⁺, and Ba²⁺, block increasing with hyperpolarization. Block by Na⁺ and Ba²⁺ was time-dependent, whereas Cs⁺ block was too fast to resolve. Rb⁺ was sparingly permeant. In cell-attached patches with high [K⁺] in the pipette, the single I _IR channel conductance was ∼30 pS and no outward current could be detected. I _BK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the conductance was ∼200–250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was observed, suggesting block by intracellular Na⁺. I _BK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship was shifted to more negative potentials by increased [Ca²⁺]. Macroscopic I _BK was blocked by external TEA⁺ with half block at 0.35 mm. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K⁺ channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold. Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed. Received: 19 September 1995/Revised: 14 March 1996     

Excitation-revealed changes in cytoplasmic Cl− concentration in “Cl−-starved”Chara cells

Summary The changes in the cytoplasmic Cl^– concentration, [Cl^–] _c , are monitored at the time of withdrawal (starvation) and subsequent replacement of Cl^– in the outside medium. The measurement technique exploits the involvement of Cl^– inChara excitation. The transient clamp current due to Cl^–,I _Cl, is separated from other excitation transients through Hodgkin-Huxley (HH) equations, which have been adjusted toChara. TheI _Cl amplitude depends on HH parameters, [Cl^–] _c and the maximum membrane conductance to Cl^–, . The results are discussed in terms of these quantities.I _Cl and were found to fall after 6–10 hr of Cl^– starvation, thus supporting the hypothesis that [Cl^– _c decreases in Cl^–-free medium. The best HH fit to starved data was obtained with [Cl^– _c =3.5mm. The time-course forI _Cl decline is considerably slower than the time-course of the rise of the starvation-stimulated influx. As cells starved for periods longer than 24 hr are re-exposed to Cl^–, it is revealed that while [Cl^–] _c remains low during long starvation, increases to values greater than those of the normal cells. Such differences among cells starved for various lengths of time have not been detected previously.     

Anaerobic contribution to the time to exhaustion at the minimal exercise intensity at which maximal oxygen uptake occurs in elite cyclists, kayakists and swimmers

Using 23 elite male athletes (8 cyclists, 7 kayakists, and 8 swimmers), the contribution of the anaerobic energy system to the time to exhaustion (t _lim) at the minimal exercise intensity (speed or power) at which maximal oxygen uptake (V˙O_{2 max}) occurs (I _{V˙O2 max}) was assessed by analysing the relationship between the t _lim and the accumulated oxygen deficit (AOD). After 10-min warming up at 60% of V˙O_{2 max}, the exercise intensity was increased so that each subject reached his I _V˙O2max in 30 s and then continued at that level until he was exhausted. Pre-tests included a continuous incremental test with 2 min steps for determining the I _V˙O2max and a series of 5-min submaximal intensities to collect the data that would allow the estimation of the energy expenditure at I _V˙O2max . The AOD for the t _lim exercise was calculated as the difference between the above estimation and the accumulated oxygen uptake. The mean percentage value of energy expenditure covered by anaerobic metabolism was 15.2 [(SD 6)%, range 8.9–24.1] with significant differences between swimmers and kayakists (16.8% vs 11.5%, P≤0.05) and cyclists and kayakists (16.4% vs 11.5%, P≤0.05). Absolute AOD values ranged from 26.4 ml · kg⁻¹ to 83.6 ml · kg⁻¹ with a mean value of 45.9 (SD 18) ml · kg⁻¹. Considering all the subjects, the t _lim was found to have a positive and significant correlation with AOD (r = 0.62, P≤0.05), and a negative and significant correlation with V˙O_{2 max} (r = −0.46, P≤0.05). The data would suggest that the contribution of anaerobic processes during exercise performed at I _V˙O2max should not be ignored when t _lim is used as a supplementary parameter to evaluate specific adaptation of athletes. Accepted: 17 December 1996     

Slow low-threshold potassium current inHelix pomatia neurons

A low-threshold outward current was studied in the neurons ofHelix pomatia at –70 to –30 mV using a two-electrode voltage clamp technique. In addition to the well-known A current (I _A), a slower outward current calledI _As (slow) was revealed. Activation and inactivation times ofI _As at –40 mV ranged from 90 to 120 msec and from 3 to 5 sec, respectively. The current recovered within 2 to 5 sec after inactivation at –120 mV. Analysis of changes in the reversal potential ofI _As caused by an increase in external potassium concentration suggests a potassium origin forI _As. The curves ofI _As stationary activation and inactivation fit the Boltzmann equation. Deriving from an activation curve, the activation potential for a half-maximum current,, is –40 mV, and the slope factor,k, is –9.8 mV, while those values for the inactivation curve are –84 mV (a half-maximum inactivation) and 7.5 mV.I _As is blocked by 4-aminopyridine (1–30 µM), tetraethylammonium (1 mM), and Ba²⁺ (1 mM), but is resistant to Cs⁺ (1 mM). PeakI _As is not affected either by substitution of external Ca²⁺ for Mg²⁺ or by application of Cd²⁺ (0.5–1.0 mM). The results suggest that activation ofI _As does not require Ca²⁺ entry into the cell.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 427–432, November–December, 1993.     

Singlet Oxygen and Other Reactive Oxygen Species are Involved in Regulation of Release of Iron-Binding Chelators from Scenedesmus cells

Freshly-added iron only slightly affected the growth of iron-sufficient cells of the green alga Scenedesmus incrassatulus Bohl, strain R-83, but induced accumulation of malondialdehyde (MDA) in cells and excretion of MDA in the medium. These effects were stronger in response to Fe²⁺ as compared to Fe³⁺, but Fe³⁺ induced the release of more iron-binding chelators from these cells than Fe²⁺. Fe³⁺ added either in dark or in light induced release of equal concentrations of iron-complexing agents, part of which formed strong chelates with iron in the medium. Exogenously added hydrogen peroxide inhibited iron-induced release of chelators but the effect was removed by addition of the hydroxyl radical scavenger dimethylsulfoxide (DMSO). Malondialdehyde also inhibited the release of chelators. Release of chelators was induced in the absence of iron salts by photoexcited chlorophyll (Chl). The Chl-induced release was efficiently inhibited by singlet oxygen scavengers such as dimethylfuran, -carotene, sodium azide and vitamin B₆, and stimulated in D₂O or DMSO. Exogenously added catalase inhibited the release more than added superoxide dismutase. The Fe³-induced release of chelators was also inhibited by scavengers of singlet oxygen, but was not affected by sodium azide and by ethanol. Hence both H₂O₂ and singlet oxygen were involved in induction of chelator release in the absence of iron in light. The induction of chelator release by iron in dark involved H₂O₂, but not singlet oxygen.     

The mechanism of Na+ transport by rabbit urinary bladder

Summary The mechanism of Na⁺ transport in rabbit urinary bladder has been studied by microelectrode techniques. Of the three layers of epithelium, the apical layer contains virtually all the transepithelial resistance. There is radial cell-to-cell coupling within this layer, but there is no detectable transverse coupling between layers. Cell coupling is apparently interrupted by intracellular injection of depolarizing current. The cell interiors are electrically negative to the bathing solutions, but the apical membrane of the apical layer depolarizes with increasingI _sc. Voltage scanning detects no current sinks at the cell junctions or elsewhere. The voltage-divider ratio, , (ratio of resistance of apical cell membrane,R _a, to basolateral cell membrane,R _b) decreases from 30 to 0.5 with increasingI _sc, because of the transportrelated conductance pathway in the apical membrane. Changes in effective transepithelial capacitance withI _sc are predicted and possibly observed. The transepithelial resistance,R _t, has been resolved intoR _a, R_b, and the junctional resistance,R _j, by four different methods: cable analysis, resistance of uncoupled cells, measurements of pairs of (R _t, ) values in the same bladder at different transport rates, and the relation betweenR _t andI _sc and between andI _sc.R _j proves to be effectively infinite (nominally 300 k F) and independent ofI _sc, andR _a decreases from 154 to 4 k F with increasingI _sc. In the resulting model of Na⁺ transport in tight epithelia, the apical membrane contains an amiloride-inhibited and Ca⁺⁺-inhibited conductance pathway for Na⁺ entry; the basolateral membrane contains a Na⁺–K⁺-activated ATPase that extrudes Na⁺; intracellular (Na⁺) may exert negative feedback on apical membrane conductance; and aldosterone acts to stimulate Na⁺ entry at the apical membrane via the amiloride-sensitive pathway.     

Simultaneous Measurement of Dissolved Oxygen and Oxidation-Reduction Potentials in the Aerobic Culture

The most dominant factor influencing the oxidation-reduction potentials (E) in the cultured system was oxygen tension. H was an useful index to express the degree of oxygen supply in place of dissolved oxygen (P_L) under a limited oxygen supply. The conversion of microbial products caused by the change in oxygen supply was clearly analyzed by the use of E value. Bacillus subtilis excreted lactic acid at the E value ?220 mV, 2,3-butyleneglycol at ?195 mV and acetoin at ?160mV as the main product. E also gave the significant information concerning the changes in cell’s respiration. Cyanide at the concentration of 10^?5m, azide at 10^?3m and 2,4-dinitrophenol (DNP) at 10^?2m inhibited cell respiration causing the decrease in E and the increase in P_L, and DNP at 0.4×10^?3m promoted oxygen uptake of the cells causing the decrease in both E and P_L.     

Cellular and shunt conductances of toad bladder epithelium

Summary Toad urinary bladders were mounted in Ussing-type chambers and voltage-clamped. At nonzero voltages only, small fluctuations in current, I, and therefore in tissue conductance, G _t, were detected. These fluctuations were caused by the smooth muscle of the underlying tissue which could be monitored continuously and simultaneously with the current,I. Inhibition of the smooth muscle contraction with verapamil (2×10^–5 m) abolished the fluctuations inI andG _t. Amiloride (10^–4 m) had no significant effect on the magnitude of G _t, oxytocin increasedG _t without affecting G _t, and mucosal hypertonicity produced by mannitol increased G _t. These results are consistent with the hypothesis that two parallel pathways exist for passive current flow across the toad urinary bladder: one, the cellular pathway, was not affected by smooth muscle activity; the other, the paracellular pathway, was the route whose conductance was altered by the action of the smooth muscle.Thus the relationship between the cellular and shunt conductances of the epithelium of the toad urinary bladder, under a variety of conditions, can be investigated by utilizing the effects of the movement of the smooth muscle.     

Changes in the kinetics of phosphate and potassium absorption in nutrient-deficient barley roots measured by a solution-depletion technique

From measurements of the rates of depletion of labelled ions from solution in the low concentration range, we described the phosphate and potassium uptake characteristics of the roots of intact barley plants in terms of the kinetic parameters, K _m and I _max (the maximum rate of uptake). In relatively young (13 d) and older (42 d) plants, cessation of phosphate supply for 4 d or more caused a marked increase in I _max (up to four times), without concomitant change in K _m, which remained between 5 and 7 M. By contrast, 1 d of potassium starvation with 14-d plants caused a decline in the K _m (i.e. an increased apparent affinity for potassium) from 53 M to 11 M, without alteration to I _max. After longer periods of potassium starvation, I _max increased (about two times) while the K _m remained at the same low value. Growth of shoots and roots were unaffected by these treatments, so that concentrations of ions in the tissues declined after 1 d or more of nutrient starvation, but we could not identify a characteristic endogenous concentration for either nutrient at which changes in kinetic parameters were invariably induced. The possible mechanisms regulating carriermediated transport, and the importance of changes induced in kinetic parameters in ion uptake from solution and soil are discussed.Symbol I _max the maximum rate of absorption at saturating concentrations     

Energetic metabolism during individual development of <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> (Lymnaeidae,gastropoda): III. Late postlarval ontogeny

Changes in the rate and intensity of oxygen consumption during individual ontogeny of 14 specimens of Lymnaea stagnalis in the period from the 10th week after emergence until death was investigated in aquaculture. It was demonstrated that the rate of oxygen consumption increased and the intensity of this process decreased during the whole period of observations. Alterations of these parameters were accompanied with permanent oscillations of their meanings. The correlation between intensity of oxygen consumption (q) and age (t) can be described with the equation q = q _st/(1-exp(−k _g (t+t ₀))). The values of coefficients of this equation do not differ significantly between individuals and, on average, comprise k _g = 0.0696 ± 0.0072 weeks⁻¹; q _st = 60.4 ± 2.6 mcl O₂/(h · g); t ₀ = −3.0 ± 0.7 weeks. The dependence of the rate of oxygen consumption (Q, μl O₂/h) on body weight (M, g) for all data is significantly described with the allometric equation Q = 0.369M ^0.779.     

Electrophysiological and Theoretical Analysis of Depolarization-Dependent Outward Currents in the Dendritic Membrane of an Identified Nonspiking Interneuron in Crayfish

Depolarization-dependent outward currents were analyzed using the single-electrode voltage clamp technique in the dendritic membrane of an identified nonspiking interneuron (LDS interneuron) in situ in the terminal abdominal ganglion of crayfish. When the membrane was depolarized by more than 20 mV from the resting potential (65.0 ± 5.7 mV), a transient outward current was observed to be followed by a sustained outward current. Pharmacological experiments revealed that these outward currents were composed of 3 distinct components. A sustained component (I _s) was activated slowly (half rise time > 5 msec) and blocked by 20 mM TEA. A transient component (I _t1) that was activated and inactivated very rapidly (peak time < 2.5 msec, half decay time < 1.2 msec) was also blocked by 20 mM TEA. Another transient component (I _t2) was blocked by 100 M 4AP, activated rapidly (peak time < 10.0 msec) and inactivated slowly (half decay time > 131.8 msec). Two-step pulse experiments have revealed that both sustained and transient components are not inactivated at the resting potential: the half-maximal inactivation was attained at –21.0 mV in I _t1, and –38.0 mV in I _t2. I _s showed no noticeable inactivation. When the membrane was initially held at the resting potential level and clamped to varying potential levels, the half-maximal activation was attained at –36.0 mV in I _s, –31.0 mV in I _t1 and –40.0 mV in I _t2. The activation and inactivation time constants were both voltage dependent. A mathematical model of the LDS interneuron was constructed based on the present electrophysiological records to simulate the dynamic interaction of outward currents during membrane depolarization. The results suggest that those membrane conductances found in this study underlie the outward rectification of the interneuron membrane as well as depolarization-dependent shaping of the excitatory synaptic potential observed in current-clamp experiments.     

Soluble or Bound Laminin Elicit in Human Neuroblastoma Cells Short- or Long-Term Potentiation of a K+ Inwardly Rectifying Current: Relevance to Neuritogenesis

Changes in the resting potential (V_rest) and in the underlying ionic conductances were measured by the patch-clamp technique in SH-SY5Y human neuroblastoma cells exposed to substrate-bound or soluble Laminin (bLN: sLN), as compared to integrin-independent substrates (polylysine (PL); bovine serum albumin (BSA)). While PL and BSA were ineffective, both forms of LN caused an early (5-15 min) activation of a peculiar type of Inwardly Rectifying K^? current (I,_ir) characterised by a voltage-dependent inactivation in the range of membrane potentials around —50/0 mV. I_ir was blocked by Cs⁺ ions and by the antiarrhythmic drug E-4031, a specific inhibitor of the HERG-codified channels. In cells adherent to bLN, I,_ir potentiation (85%) persisted for 90-120 min and was accompanied by a similar, but transient, increase in the leakage conductance (G_l). Successively, the persistence of a high I_ir conductance and the decrease of G_l progressively bring V_rest from –12 to -30 mV in about 120 min. On the other hand, in cells adherent to PL, exposure to sLN produced a similar but not persistent activation of I_ir, without any increase in G_l: this caused a rapid, transient hyperpolarisation of V_rest The effects of bLN and sLN were mimicked by antibodies raised against the integrin β₁ subunit, suggesting a specific integrin-mediated mechanism. In fact, when bound to the culture dishes, these antibodies simultaneously activated the I_ir and G_l, whereas in soluble form they only activated I_ir. Cells adherent to bLN emitted neurites, a process impaired by the block of I_ir by E-4031 and Cs⁺. On the whole data suggest that the integrin-mediated activation of I_ir plays a crucial role in the commitment to neuritogenesis of neuroblastoma cells, independently on the effects of this activation on V_rest.