Inhibiteurs anti-trypsine et activités protéolytiques des Albumines de graine de Vigna unguiculata

Summary The extracellular space of tentacles of Drosera capensis L. is divided into two compartments by cuticular material between cells of an endodermoid layer and by the nonporous cuticle of the stalk and neck. The distal compartment includes the mucilaginous secretion as well as the free space of the secretory cap, since the cuticle covering the cap is perforated by numerous 0.05–0.3 m pores. The proximal compartment includes xylem and the intercellular space of the stalk. The existence of the endodermoid partition is consistent with the observation that action potentials recorded extracellularly from the head may be positive-going while those recorded extracellularly from the stalk are negative-going. The partitioning is also consistent with the hypothesis previously proposed to explain why the amplitude of action potentials recorded from the mucilage varies as a function of the amplitude of the receptor potential.The living cells are united by plasmodesmata. Unusually abundant plasmodesmata were observed in the walls between endodermoid cells and neck cells, between neck cells and the next row of outer stalk cells, in the end walls connecting the outer stalk cells, and the end walls connecting the inner stalk cells: these strategically located plasmodesmata presumably permit the electrotonic spread of receptor potentials and action potentials between cells.     

Increase in the levels of activity of polyadenylic acid-metabolizing enzymes following phytohaemagglutinin stimulation of human lymphocytes

Increased levels of soluble activity of all three enzymes involved in polyadenylic acid metabolism were measured in PHA-stimulated versus normal lymphocytes. Poly(A)-polymerase and poly(A)-exonuclease values increased significantly (from 25.7 ± 4.2 (S.E.M.) to 53.5 ± 10.6 (S.E.M.), and from 334.6 ± 33.2 (S.E.M.) to 653.2 ± 53.4 (S.E. M.) respectively), while a moderate increase was observed in poly(A)-endonuclease (from 299.2 ± 33.8 (S.E.M.) to 403.0 ± 77.1 (S.E.M.). The above differences persisted after two fractionations of the crude cell extracts by ion exchange chromatography and molecular sieving, and could not be attributed to the competitive action of all three enzymes in the untreated extracts. Fractionation of the extracts of resting and stimulated cells on Sephadex G-75 revealed two molecular forms of poly(A)-polymerase activity.Abbreviations poly(A) or An Polyadenylic acid - oligo(A) or A₁₀ oligoadenylic acid - poly(C) polycytidylic acid - poly(U) polycytidylic acid - poly(G) polyguanylic acid - poly(dA) polydeoxyadenylic acid - EDTA ethylenediamine tetraacetate - PHA phytohaemagglutinin - PBS phosphate buffered saline - NP-40 nonidet-40 - KPi buffer potassium phosphate buffer     

Volume flows and pressure changes during an action potential in cells ofChara australis

Summary Methods have been used for monitoring either volume flows or pressure changes, simultaneously with membrane potentials, in giant algal cells ofChara australis during an action potential. The volume flows were measured from the movement of a mercury bead in a capillary tube recorded by a photo-transducer. The pressure changes were measured by monitoring the deflection of a thin wedge, resting transversely across a cell, and using the same photo-transducer, the deflection of the wedge being directly related to the cell's turgor pressure. The average maximum rate of volume flow per unit area during an action potential was 0.88±0.11 nliter·sec^–1·cm^–2 in the direction of an outflow from the cell (total volume outflow being about 3 nliter·cm^–2 per action potential). Similarly, the maximum rate of change of pressure was 19.6±3.8×10^–3 atm·sec^–1 (peak change being 19.3±2.9×10^–3 atm equivalent to 14.7±2.2 mm Hg). The volume flow and pressure changes followed the vacuolar potential quite closely, the peak rate of volume flow lagging behind the peak of the action potential by 0.17±0.08 sec and the peak rate of pressure change leading it by 0.09±0.07 sec.     

Membrane properties of identified embryonic nerve and glial cells of the leech central nervous system

Summary The membrane potential of identified nerve (Retzius) cells and neuropil glial cells from 11 (±1) day-old embryos of the leechHirudo medicinalis was recorded using conventional intracellular microelectrodes. At this stage all ganglia of the segmental nervous system are formed. The membrane potential of Retzius cells was –68±4 mV (±SD,n=8), and showed a slope of 42 mV between 10 mM and 100 mM external K concentration. Retzius cells were able to fire action potentials which had a fast Na-dependent component, and, under appropriate conditions, also generated slow Ca (Ba) action potentials. The mean membrane potential of the neuropil glial cell at physiological K concentration (4 mM) was –83±5 mV (±SD,n=10), and showed a dependence of 56 mV for a tenfold change in the external K concentration (> 4mM). Neuropil glial cells showed no signs of voltage-activated excitability, but they repeatedly depolarized in the presence of 0.1 mM 5-HT.     

Electrical rhythmicity and spread of action potentials in longitudinal muscle of guinea pig distal colon

Using simultaneous intracellular recordings, we have characterized 1) electrical activity in the longitudinal muscle (LM) of isolated segments of guinea pig distal colon free to contract spontaneously and 2) extent of propagation of spontaneous action potentials around the circumference of the colon. In all animals, rhythmical spontaneous depolarizations (SDs) were recorded that are usually associated with the generation of action potentials. Recordings from pairs of LM cells, separated by 100 microm in the circumferential axis, revealed that each action potential was phase locked at the two electrodes (mean propagation velocity: 3 mm/s). However, at an increased electrode separation distance of 1 mm circumferentially, action potentials and SDs became increasingly uncoordinated at the two recording sites. No SDs or action potentials ever propagated from one circumferential edge to the other (i.e., 13 mm apart). When LM strips were separated from the myenteric plexus and circular muscle, rhythmically firing SDs and action potentials were still recorded. Atropine (1 microM) or tetrodotoxin (1 microM) either reduced the frequency of SDs or temporarily abolished activity, whereas nifedipine (1 microM) always abolished SDs and action potentials. Kit-positive interstitial cells of Cajal were present at the level of the myenteric plexus and circular and longitudinal muscle. In summary, SDs and action potentials in LM propagate over discrete localized zones, usually <1 mm around the circumference of the colon. Furthermore, in contrast to the classic slow wave, rhythmic depolarizations in LM appear to be generated by an intrinsic property of the smooth muscle itself and are critically dependent on opening of L-type Ca(2+) channels.     

Receptor potentials and action potentials in Drosera tentacles

Summary Voltage fluctuations identified as receptor potentials can be detected with electrodes applied to the mucilage surrounding the head of a tentacle of Drosera intermedia if the head is stimulated by contact with a live insect, by the touch of a clean, inert object, or by application of salt solutions. Associated with a low receptor potential are action potentials, which occur at a frequency dependent on the magnitude of the receptor potential. These action potentials can be detected with electrodes applied to any region of the stalk of the tentacle. Inflection of the lower stalk follows the occurrence of action potentials. Inflection is minute for isolated action potentials but large and rapid when several occur within a brief interval.The apparent amplitude of action potentials recorded from the stalk is independent of receptor potential amplitude, but that of action potentials recorded from the mucilage commonly decreases as the receptor potential deviates from the baseline and increases as it returns. It is suggested that variation of apparent amplitude of the action potentials may result from postulated variation in the resistance of receptor membranes.     

Characteristics of cardiac action potentials in marsupials

Summary Standard microelectrode techniques were used to record action potentials from single atrial, ventricular and Purkinje fibers of hearts taken from three species of marsupial (Macropus rufus, Macropus robustus andMacropus eugenii) and from dogs, sheep and guinea-pigs. The major electrophysiological parameters of marsupial potentials were qualitatively similar to the values for placental mammals. The grouped data for ventricular action potentials from studies on 6 adult male red kangaroos (Macropus rufus) were (mean ±SD): Resting potential –69.5±5.0 mV; action potential amplitude 92.7±5.7 mV; action potential duration (to 90% repolarization): 182.5±17.5 ms; maximum rate of depolarization: 196.5±80.1 V/s. The major point of difference was the short duration of the red kangaroo ventricular action potential compared to those of the placental mammals, and compared to atrial cells from the kangaroos. It is suggested that this explains the short QT interval reported by others for kangaroo electrocardiograms, and that it may also be implicated in the high frequency of sudden death previously noted in these animals.     

Reciprocal changes in center and surrounding S potentials of fish retina in response to dopamine

Effects of dopamine (DA) were examined on the intracellularly recorded potential from horizontal cells in the fish (Eugerres plumieri) retina. DA (100 M in the perfusate) augmented the center S potential in a response to a spot illumination and attenuated the surrounding S potential to an annular light by approximately 40%. These reciprocal changes in the S potentials were associated with a slight depolarization (2.5 mV) of the horizontal cell, and were reversible in 10–15 min. The results indicate that DA at this concentration does not affect directly the synaptic transmission from photoreceptors to horizontal cells, while it appears to interfere selectively with the lateral propagation of an S potential. The effects of DA observed may represent an aspect of function of DA-containing interplexiform cells in the retina.     

Influence of cell cycle phases on the electrical activity and hormone release in a transformed line of anterior pituitary cells

Electrophysiological experiments have shown that about 50% of cultured GH3 cells (tumoral cell line, from the anterior pituitary gland) are inexcitable i.e. they do not display action potentials either spontaneously or when depolarized by a current pulse. We report here this inexcitability may be related to cellular kinetics. Thus we have studied the relationship between the various phases of the cell cycle, the electrophysiological properties of GH3/B6 cells and spontaneous or induced Prolactin and Growth Hormone (GH) release rates. Asynchronous populations of viable cells were stained with Hoechst 33 342 DNA fluorescent dye, and sorted using a flow cytometer into G1 and S phases. After selection intracellular potentials were recorded using a single glass micro-electrode; the basal or TRH stimulated rates of PRL and GH secretions were determined by RIA. Electrical properties of the cells i.e. resting potentials, input membrane resistance and excitability, reached a maximum for cells in G2+M phases. Only cells in G2+M displayed action potentials and TRH increased their secretion by 5 times for GH and by 6 times for PRL. In G1 and S phases the cells were electrically inactive and secretion rates remained at their basal levels. These findings demonstrate that the mechanism of stimulus secretion coupling is dependent upon the phases of the cell cycle.     

Properties of action potentials in Drosera tentacles

Summary Action potentials of Drosera tentacles resemble those of vertebrate peripheral nerves in that they appear to be comprised of relatively uniform spikes, variable shoulders or negative after-potentials, and variable positive after-potentials. The peaking of the spike corresponds to a period of great refractoriness, while action potentials of low amplitude may be fired readily during the negative after-potential. The action potentials fired during the negative after-potential appear to be unlike those of peripheral nerves in that they are of abnormally brief duration. Also apparently different from the case in peripheral nerves is the dependence of the duration of an action potential on the interval separating it from the preceding action potential.Action potentials propagate from the neck of the stalk to its base at about 5 mm s^-1 at room temperature. Propagation may be reversed artificially, consistent with the possibility that the neuroid cells are electrically coupled.     

Sodium currents in toad cardiac pacemaker cells

Cells in the pacemaker region of toad (Bufo marinus) sinus venosus had spontaneous rhythmic action potentials. The rate of firing of action potentials, the rate of diastolic depolarization and the maximum rate of rise of action potentials were reduced by TTX (10 nm to 1 m). Currents were recorded with the whole cell, tight seal technique from cells enzymatically dissociated from this region. Cells studied were identified as pacemaker cells by their characteristic morphology, spontaneous rhythmic action potential activity that could be blocked by cobalt but not by TTX and lack of inward rectification. When calcium, potassium and nonselective cation currents (I_f) activated by hyperpolarization were blocked, depolarization was seen to generate transient and persistent inward currents. Both were sodium currents: they were abolished by tetrodotoxin (10 to 100 nm), their reversal potential was close to the sodium equilibrium potential and their amplitude and reversal potential were influenced as expected for sodium currents when extracellular sodium ions were replaced with choline ions. The transient sodium current was activated at potentials more positive than –40 mV while the persistent sodium current was obvious at more negative potentials. It was concluded that, in toad pacemaker cells, TTX-sensitive sodium currents contributing both to the upstroke of action potentials and to diastolic depolarization may play an important role in setting heart rate.We thank the Australian National Heart Foundation for their support. D.A.S. is an NHMRC Senior Research Officer.     

Spontaneous Action Potential Initiation and Propagation in Regenerating Cell Segments of Acetabularia mediterranea

Thavarungkul, P., Lertsithichai, S. and Sherlock, R. A. 1987.Spontaneous action potential initiation and propagation in regeneratingcell segments of Acetabularia mediterranea.—J. exp. Bot.38: 1541–1556. The spontaneous action potentials which occur during the regenerationof anucleate isolated stalk segments (ISS's) of the unicellularalga Acetabulana mediterranea have been studied using a novelnon-invasive technique. This involved measurement of spatialsamples of the time dependent potential in the external medium(sea water) and an inverse transformation to give the currentdistribution at the cell surface. The initiation region andsubsequent propagation (if any) of each action potential wasthen analysed from the computed transmembrane currents. Theresults showed that the occurrences of the spontaneous actionpotentials followed a rhythm which had a period of approximately24–30 h. These action potentials initiated more frequentlyat the ends of the ISS's than in the middle region. Our resultsshowed no firm correlation between the regions of action potentialinitiation and the site of the regenerating apex. Generallyboth propagating and non-propagating action potentials wereobserved in the same ISS, the ratio of which seems to dependon the length of the cell. The analysed initiation sites andthe propagating behaviour of the spontaneous action potentialgenerated by the ISS's during the regeneration process do notprovide any clear support to the symmetry breaking role of thepropagating action potentials as proposed by some workers. Key words: Acetabularia, regeneration, action potential     

Pre- and post-synaptic actions of the cerebral ventral giant cell on buccal muscles ofPhiline

Summary The cerebral ventral giant cell ofPhiline exterts a selective presynaptic inhibitory modulatory action on the terminals of a buccal excitatory motoneuron in two buccal muscles. Other excitatory inputs to the muscles are not affected. The ventral giant cell also makes direct synaptic contacts on the fibres of the same muscles. In the retractor muscle M4, 5 the junction potentials are usually depolarising when measured in sea water, but in the fibres of M6 they may have either polarity. The mean membrane potential of the fibres of M4, 5 and M6 was –74.7±0.65 mV and –64±0.95 mV respectively. Depolarization of the muscles fibres by around 15 mV by immersion in 20 mM K saline abolished the junction potential in M4, 5 and converted the depolarizing potential in M6 to a hyperpolarizing response.It is concluded that the VGC junction potential results from an increase in membrane conductance to an ion with a reversal potential between –60 and –70 mV.     

The Effects of Temperature on Action Potentials in the Chill Sensitive Seismonastic Plant Biophytum sensitivum

The seismonastic plant Biophytum sensitivum was stimulated electricallyto produce reproducible action potentials. It was found thatnegative voltages only induced a response and that stimulationcould also be achieved by electrostatic means by depositingnegative charge on the leaf surface. A study of the effectsof temperature on the propagation rates of action potentialsin different aged leaves showed that for leaves of all agesthe fastest propagation occurred at 24 ?C. As the temperaturewas decreased or increased from 24 ?C a reduction in the propagationrate occurred. No propagation was found at, or below, 14 ?Cand the propagation rate decreased with increasing leaf age.Both the threshold voltage level and the charge required toproduce action potentials in young leaves was lowest at 24 ?Cand had to be increased at either lower or higher temperaturesto cause leaf closure. The duration of the electrical stimulusrequired to induce action potentials was found to decrease asthe voltage was increased. Mechanisms which could account forthe effects of temperature on action potentials are discussedin relation to the chill-sensitivity of the plant. Key words: Chilling, Action Potentials, Biophytum sensitivum     

Influence of culture passages on growth kinetics and adenovirus vector production for gene therapy in monolayer and suspension cultures of HEK 293 cells

To characterize the changes in cell growth rate and adenovirus vector (AdV) production capability of 293 cells during culture passages, 293 cells obtained at the 31st culture passage from ATCC (293M #31) were maintained as a monolayer culture and 293 cells obtained at an unknown culture passage from Invitrogen (293S) were maintained as suspension culture. In monolayer culture, the specific growth rate () of 293M cells increased rapidly with culture passage up to passage 65 and thereafter became saturated. The of 293M passage 43 (#43) was 0.29 day^–1, while the average of 293M from #66 to #86 was 0.74±0.01 day^–1 (average ± standard deviation). It was also noted that the cells became smaller in size during early culture passages. AdV production was also influenced by the number of culture passages. The AdV titer in the culture of 293M #66 was ca. tenfold higher than that of 293M #44, resulting from both a higher cell concentration and a higher AdV titer per cell at #66. In contrast, the , cell size, and AdV production of 293S cells in suspension culture did not change significantly as the culture passage number increased up to #40. Taken together, the culture passage influenced cell growth and AdV production of 293M cells in monolayer culture, but not those of 293S cells in suspension culture.     

PKC independent inhibition of voltage gated calcium channels by volatile anesthetics in freshly isolated vascular myocytes from the aorta

In this study we used barium currents through voltage gated L-type calcium channels (recorded in freshly isolated cells with a conventional patch-clamp technique) to elucidate the cellular action mechanism for volatile anesthetics. It was found that halothane and isoflurane inhibited (dose-dependently and voltage independently) Ba²⁺ currents through voltage gated Ca²⁺ channels. Half maximal inhibitions occurred at 0.64 ± 0.07 mM and 0.86 ± 0.1 mM. The Hill slope value was 2 for both volatile anesthetics, suggesting the presence of more than one interaction site. Current inhibition by volatile anesthetics was prominent over the whole voltage range without changes in the peak of the current voltage relationship. Intracellular infusion of the GDPβS (100 μM) together with staurosporine (200 nM) did not prevent the inhibitory effect of volatile anesthetics. Unlike pharmacological Ca²⁺ channel blockers, volatile anesthetics blocked Ca²⁺ channel currents at resting membrane potentials. In other words, halothane and isoflurane induced an ‘initial block’. After the first 4–7 control pulses, the cells were left unstimulated and anesthetics were applied. The first depolarization after the pause evoked a Ca²⁺ channel current whose amplitude was reduced to 41 ± 3.4% and to 57 ± 4.2% of control values. In an analysis of the steady-state inactivation curve for voltage dependence, volatile anesthetics induced a negative shift of the 50% inactivation of the calcium channels. By contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unitary L-type Ca²⁺ channels blockade occurred under cell-attached configuration, suggesting a possible action of volatile anesthetics from within the intracellular space or from the part of the channel inside the lipid bilayer.     

Comparison of the duration and power spectral changes of monopolar and bipolar M waves caused by alterations in muscle fibre conduction velocity

The muscle compound action potential (M wave) recorded under monopolar configuration reflects both the propagation of the action potentials along the muscle fibres and their extinction at the tendon. M waves recorded under a bipolar configuration contain less cross talk and noise than monopolar M waves, but they do not contain the entire informative content of the propagating potential. The objective of this study was to compare the effect of changes in muscle fibre conduction velocity (MFCV) on monopolar and bipolar M waves and how this effect depends on the distance between the recording electrodes and tendon. The study was based on a simulation approach and on an experimental investigation of the characteristics of surface M waves evoked in the vastus lateralis during 4-s step-wise isometric contractions in knee extension at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% MVC. The peak-to-peak duration (Durpp) and median frequency (Fmedian) of the M waves were calculated. For monopolar M waves, changes in Durpp and Fmedian produced by MFCV depended on the distance from the electrode to the tendon, whereas, for bipolar M waves, changes in Durpp and Fmedian were largely independent of the electrode-to-tendon distance. When the distance between the detection point and tendon lay between approximately 15 and 40 mm, changes in Durpp of bipolar M waves were more pronounced than those of distal monopolar M waves but less marked than those of proximal monopolar M waves, and the opposite occurred for Fmedian. Since, for bipolar M waves, changes in duration and power spectral features produced by alterations in MFCV are not influenced by the electrode-to-tendon distance, the bipolar electrode configuration is a preferable choice over monopolar arrangements to estimate changes in conduction velocity.     

Evoked Activity in Nerve Trunks of the Rat: 4-Aminopyridine-Induced Modifications

In experiments on rats, we studied 4-aminopyridine (4-AP)-induced modifications of the excitability of peripheral nerve fibers in an efferent trunk, the ventral root (VR), and in a mixed trunk including both afferent and efferent fibers, the sciatic nerve (SN). For this purpose, we examined how 4-AP influenced the parameters of integral action potentials recorded from the VR and SN in three experimental modes. These were: (i) stimulation of the SN and recording of antidromic action potentials from the VR in vivo after systemic injections of 4-AP into the animal, (ii) stimulation of a preparation of the SN dissected from the animal after systemic injection of 4-AP and recording of action potentials from another segment of the same preparation in vitro, and (iii) stimulation of an SN preparation and recording of action potentials from another region of this preparation in vitro, but after direct application of the solution of 4-AP to this preparation. It was found that 4-AP significantly increased the threshold for generation of action potentials and enhanced their amplitude, decreased the duration of action potentials recorded from the VR, and shortened the refractory period following these responses. The drug also significantly increased the amplitude and decreased the duration of action potentials recorded from the SN in vitro after systemic injections of the agent, but the threshold for response generation in this preparation noticeably dropped; the post-response refractory period in this case showed no changes. Modifications of action potentials recorded from the SN in vitro after direct applications of 4-AP were in general similar to the described above. Other examined parameters of action potentials (chronaxia and dynamics of an increase in the amplitude related to intensification of stimulation) showed no significant changes under the influence of 4-AP. We conclude that 4-AP increases the excitability of nerve fibers in the nerve trunks under study, but not to the point where the electrical interaction between excited and nonexcited fibers in the fiber conductors under study (VR and SN) overcomes the threshold.     

Development, survival and fecundity of the urine fly, Scatella (Teichomyza) fusca and predation by the black dumpfly, Hydrotaea aenescens

Development, survival and fecundity for Scatella (Teichomyza) fusca Maquart (Diptera: Ephydridae) were studied at 20 ± 1 °C and 85 ± 10% r.h. Mean (± S.E.) developmental times of eggs, larvae and pupae were 4.0 ± 0.06, 14.9 ± 0.19 and 14.6 ± 0.11 days, respectively, the mean (± S.E.) survival of the original egg cohort to the start of larval, pupal and adult stage being 77.2 ± 3.2%, 54.5 ± 2.6% and 47.9 ± 3.5%, respectively. Females and males displayed approximately straight survivorship curves during adult life, implying constant mortality rates. Mean (± S.E.) adult longevity was 41.6 ± 2.98 days for females and 51.2 ± 3.91 days for males. Assuming a stable age distribution the population consisted of 56% eggs, 31% larvae, 6% pupae and 7% adults. Oviposition peaked when females were 25 days old, and the highest reproductive values (RV_x) (mean ± S.E.) ranged from 129.1 ± 7.57 to 138.5 ± 6.83 for individuals 17–27 days old. A female deposited a mean (± S.E.) of 614.7 ± 35.9 female eggs over a maximum life span of 93 days. The basic reproductive rate (R ₀) (mean ± S.E.) was 173.0 ± 14.2 female offspring per female and the intrinsic rate of natural increase of female individuals (r) (mean ± S.E.) was 0.088 ± 0.001 day^-1. The mean (± S.E.) generation time (T) was 57.8 ± 0.78 days. In cultures with equal numbers of first instar S. fusca larvae and predacious third instar larvae of Hydrotaea aenescens (synonymous Ophyra aenescens) Wiedemann, mean (± S.E.) survival to the adult stage of S. fusca (16.7 ± 8.8%) was significantly lower than in controls with S. fusca alone (58.3 ± 7.4%). The potential significance of predation by H. aenescens on S. fusca in pig farms is discussed.     

Gap junctions in excitable cells

Gap junction channels are an integral part of the conduction or propagation of an action potential from cell to cell. Gap junctions have rather unique gating and permeability properties which permit the movement of molecules from cell to cell. These molecules may not be directly linked to action potentials but can alter nonjunctional processes within cells, which in turn can affect conduction velocity. The data described in this review reveal that, for the majority of excitable cells, there are two limiting factors, with respect to gap junctions, that affect the conduction/propagation of action potentials. These are (1) the total number of channels and (2) the selective permeability of the channels. Interestingly, voltage dependence and the time course of voltage inactivation (kinetics) are not rate limiting steps under normal physiological conditions for any of the connexins studied so far. Only specialized rectifying electrical synapses utilize strong voltage dependence and rapid kinetics to permit or deny the continued propagation of an action potential.