Transport systems of Ventricaria ventricosa: asymmetry of the hyper- and hypotonic regulation mechanisms.

Hyper- and hypotonic stresses elicit apparently symmetrical responses in the alga Ventricaria. With hypertonic stress, membrane potential difference (PD) between the vacuole and the external medium becomes more positive, conductance at positive PDs (Gmpos) increases and KCl is actively taken up to increase turgor. With hypotonic stress, the membrane PD becomes more negative, conductance at negative PDs (Gmneg) increases and KCl is lost to decrease turgor. We used inhibitors that affect active transport to determine whether agents that inhibit the K(+) pump and hypertonic regulation also inhibit hypotonic regulatory responses. Cells whose turgor pressure was held low by the pressure probe (turgor-clamped) exhibited the same response as cells challenged by hyperosmotic medium, although the response was maintained longer than in osmotically challenged cells, which regulate turgor. The role of active K(+) transport was confirmed by the effects of decreased light, dichlorophenyldimethyl urea and diethylstilbestrol, which induced a uniformly low conductance (quiet state). Cells clamped to high turgor exhibited the same response as cells challenged by hypo-osmotic medium, but the response was similarly transient, making effects of inhibitors hard to determine. Unlike clamped cells, cells challenged by hypo-osmotic medium responded to inhibitors with rapid, transient, negative-going PDs, with decreased Gmneg and increased Gmpos (linearized I-V), achieving the quiet state as PD recovered. These changes are different from those exerted on the pump state, indicating that different transport systems are responsible for turgor regulation in the two cases.

The role of ultrasound in the management of flexor tendon injuries.

The use of ultrasound scanning to establish tendon pathologies was assessed retrospectively in 17 patients in 18 digits. The ultrasound scan demonstrated four patterns: (1) normal intact tendons in four, (2) ruptured tendons in three, (3) tendons in continuity but attenuated in five and (4) tendons in continuity but thickened with fibrosis and decreased movement representing adhesions in five patients. Surgery was undertaken in only three cases, confirming the ultrasound diagnosis in two. Surgery was offered to all three patients with ruptures but was declined by two. Ultrasound imaging helped to avoid surgery in 14 cases by excluding flexor tendon re-ruptures. This allowed on-going mobilisation, leading to recovery of function.

Electrophysiology of turgor regulation in marine siphonous green algae.

We review electrophysiological measures of turgor regulation in some siphonous green algae, primarily the giant-celled marine algae, Valonia and Ventricaria, with particular comparison to the well studied charophyte algae Chara and Lamprothamnium. The siphonous green algae have a less negative plasma membrane potential, and are unlikely to have a proton-based chemiosmotic transport system, dominated by active electrogenic K(+) uptake. We also make note of the unusual cellular structure of the siphonous green algae. Hypertonic stress, due to increased external osmotic pressure, is accompanied by positive-going potential difference (PD), increase in conductance, and slow turgor regulation. The relationship between these is not yet resolved, but may involve changes in K(+ )conductance (G (K)) or active K(+) transport at both membranes. Hypotonic turgor regulation, in response to decreased external osmotic pressure, is approximately 3 times faster than hypertonic turgor regulation. It is accompanied by a negative-going PD, although conductance also increases. The conductance increase and the magnitude of the PD change are strongly correlated with the magnitude of hypotonic stress.

When is a cell not a cell? A theory relating coenocytic structure to the unusual electrophysiology of Ventricaria ventricosa (Valonia ventricosa).

Ventricaria ventricosa and its relatives have intrigued cell biologists and electrophysiologists for over a hundred years. Historically, electrophysiologists have regarded V. ventricosa as a large single plant cell with unusual characteristics including a small and positive vacuole-to-outside membrane potential difference. However, V. ventricosa has a coenocytic construction, with an alveolate cytoplasm interpenetrated by a complex vacuole containing sulphated polysaccharides. We present a theory relating the coenocytic structure to the unusual electrophysiology of V. ventricosa. The alveolate cytoplasm of V. ventricosa consists of a collective of uninucleate cytoplasmic domains interconnected by fine cytoplasmic strands containing microtubules. The cytoplasm is capable of disassociating into single cytoplasmic domains or aggregations of domains that can regenerate new coenocytes. The cytoplasmic domains are enclosed by outer (apical) and inner (basolateral) faces of a communal membrane with polarised K(+)-transporting functions, stabilised by microtubules and resembling a tissue such as a polarised epithelium. There is evidence for membrane trafficking through endocytosis and exocytosis and so "plasmalemma" and "tonoplast" do not have fixed identities. Intra- and extracellular polysaccharide mucilage has effects on electrophysiology through reducing the activity of water and through ion exchange. The vacuole-to-outside potential difference, at which the cell membrane conductance is maximal, reverses its sign from positive under hypertonic conditions to negative under hypotonic conditions. The marked mirror symmetry of the characteristics of current as a function of voltage and conductance as a function of voltage is interpreted as a feature of the communal membrane with polarised K(+) transport. The complex inhomogeneous structure of the cytoplasm places in doubt previous measurements of cytoplasm-to-outside potential difference.

The different characteristics of Dupuytren's disease fibroblasts derived from either nodule or cord: expression of alpha-smooth muscle actin and the response to stimulation by TGF-beta1.

Mechanisms behind the onset and progression of Dupuytren's disease are poorly understood. Both myofibroblasts and transforming growth factor beta 1 (TGF-beta(1)) have been implicated. We studied fibroblast cultures derived from nodules or cords of Dupuytren's contracture tissue to determine the proportion of myofibroblasts present in comparison with flexor retinaculum fibroblast cultures. We identified myofibroblasts by immunohistochemical staining for alpha-SMA. We then investigated the effects of TGF-beta(1) stimulation on these fibroblasts. Basal myofibroblast/fibroblast proportions were 9.7% in nodule cell cultures, 2.7% in cord cell cultures and only 1.3% in flexor retinaculum cell cultures. Nodule and cord myofibroblast proportions increased to 25.4% and 24.2%, respectively, in response to TGF-beta(1) treatment. Flexor retinaculum cell cultures showed no response to TGF-beta(1) stimulation. Fibroblasts cultured from specific regions of Dupuytren's tissue retain myofibroblast features in culture. TGF-beta(1) stimulation causes an increased myofibroblast phenotype to similar levels in both nodule and cord, suggesting that previously quiescent cord fibroblasts can be reactivated to become myofibroblasts by TGF-beta(1). This could be an underlying reason for high recurrence rates seen after surgery or progression following injury.

The transport systems of Ventricaria ventricosa: hypotonic and hypertonic turgor regulation.

The time course of hypertonic and hypotonic turgor regulation was studied in Ventricaria (Valonia) using pressure probe and I/V(current-voltage) analysis. Of 11 cells, 9 exhibited hypertonic turgor regulation, ranging from 100% regulation in 150 min to 14% regulation (14% recovery of the decrease in turgor) in 314 min. Some cells began regulating immediately, others took up to 90 min to begin. The resting PD (potential difference) became more positive in most cells. The I/V characteristics became more nonlinear with high resistance between -150 and -20 mV and negative conductance region near -70 mV. Prolonged (16 sec) voltage clamps to negative levels (-100 to -150 mV) showed progressively more rapid current turn-off, but subsequent I/V characteristics were not affected. Clamping to +150 mV, however, abolished the high conductance between -50 and +100 mV to yield a uniform high resistance I/V characteristic, similar to that in high [K+]o. Decreasing illumination from 2.02 micromol sec(-1) m(-2) to 0.5 micromol sec(-1)1 m(-2) had a similar effect. Two out of a total of three cells exhibited hypotonic turgor regulation. Both cells started regulating within minutes and achieved near 50% regulation within 50 min. The PD became more negative. The I/V curves exhibited high resistance between +50 and +150 mV. The characteristics were similar to those in cells exposed to low [K+]o. Prolonged voltage clamps to both negative and positive levels showed slow current increase. Decreased illumination increased the membrane resistance.

Acetylcholine-activated Cl- channel in the Chara tonoplast.

Acetylcholine has long been suggested to play a role in controlling physiological processes in plants, but no mechanism has been shown for its action. We show here that a chloride channel in the tonoplast (vacuolar membrane) of Chara corallina responds to acetylcholine. The channel has a conductance of 45 pS. The effect of acetylcholine is enhanced by nicotine, with the open probability increasing from 0.05 in the presence of 4 mM acetylcholine to 0.3 in the presence of 4 mM acetylcholine + 6 mM nicotine. Some effects of acetylcholine were seen at concentrations as low as 20 microM, with a maximum effect between 1 and 10 mM. In the intact cell, acetylcholine prolongs the depolarized phase of the action potential. We propose that this acetylcholine-gated channel has evolved separately from the mammalian acetylcholine-gated channel, and suggest that this represents a third form of acetylcholine signal transduction, after the nicotinic and muscarinic pathways in animal systems.

Do plastic surgeons resect basal cell carcinomas too widely? A prospective study comparing surgical and histological margins.

Basal cell carcinoma is a common condition facing the plastic surgeon. When formally excised, a surrounding margin of normal skin is included in an attempt to ensure complete excision. We set out to investigate our excision margins in a prospective study of 100 basal cell carcinomas in 86 patients treated by conventional surgical excision. The edge of each lesion was delineated, an excision margin was drawn, and the closest point was identified and measured. The tumours were excised, and the specimens were examined to determine the closest histological margin. A comparison was made between the marked surgical margins and the margins observed on microscopy. The mean observed surgical margin was 3.0 mm and the mean histological margin was 3.7 mm; 44% of the margins agreed to within 1 mm, 79% to within 2 mm and 92% to within 3 mm. There were four incomplete excisions, all at the lateral margin. There was agreement in the position of the closest margin in 69% of cases. The measured surgical excision margins correlated well with those assessed histologically, as did the position of the closest margin. Given a 3 mm margin, 96% of lesions would have been excised completely. We feel that our current practice represents a satisfactory balance between maintaining a low rate of incomplete excision and minimising the sacrifice of normal skin.

Long-term results of facial rejuvenation by carbon dioxide laser resurfacing using a quantitative method of assessment.

Carbon dioxide laser resurfacing has been widely reported as an effective method for treating facial rhytides. Few published series, however, have assessed the long-term results of this procedure. We prospectively reviewed our long-term results of perioral CO(2) laser resurfacing using an accurate, reproducible and quantitative method of evaluation. Wrinkle depth was measured using a silicone elastomer to provide an exact negative-replica mask of the perioral region. Simple light microscopy was then used to measure the depth of rhytides. This was carried out preoperatively, 6 weeks postoperatively and 2 years postoperatively, and the results were compared for 31 patients. CO(2) laser resurfacing achieved a highly significant mean reduction in wrinkle depth at 6 weeks of 91% (paired t -test:P <0.00001), and this was well maintained at 2 years, when the mean reduction in wrinkle depth was 87% (paired t-test: P<0.00001). During the long follow-up, complications were few, with transient erythema being the most common; there were no cases of scarring. As in other series that have reported longer follow-up, we encountered three cases of minor hypopigmentation. We have used light microscopy on silicone moulds as an accurate method of assessing outcome after laser resurfacing of perioral rhytides. The early highly significant reduction in wrinkle depth was maintained at 2 years with minimal associated short-term or long-term morbidity.

Transport systems of Ventricaria ventricosa: I/V analysis of both membranes in series as a function of [K(+)](o).

The current-voltage (I/V) profiles of Ventricaria (formerly Valonia) membranes were measured at a range of external potassium concentrations, [K(+)](o), from 0.1 to 100 mm. The conductance-voltage (G/V) characteristics were computed to facilitate better resolution of the profile change with time after exposure to different [K(+)](o). The resistance-voltage (R/V) characteristics were computed to attempt resolution of plasmalemma and tonoplast. Four basic electrophysiological stages emerged: (1) Uniform low resistance between -60 and +60 mV after the cell impalement. (2) High resistance between +50 and +150 for [K(+)](o) from 0.1 to 1.0 mm and hypotonic media. (3) High resistance between -150 and -20 mV for [K(+)](o) of 10 mm (close to natural seawater) and hypertonic media. (4) High resistance between -150 and +170 mV at [K(+)](o) of 100 mm. The changes between these states were slow, requiring minutes to hours and sometimes exhibiting spontaneous oscillations of the membrane p.d. (potential difference). Our analysis of the I/V data supports a previous hypothesis, that Ventricaria tonoplast is the more resistive membrane containing a pump, which transports K(+) into the vacuole to regulate turgor. We associate state (1) with the plasmalemma conductance being dominant and the K(+) pump at the tonoplast short-circuited probably by a K(+) channel, state (2) with the K(+) pump "off" or short-circuited at p.d.s more negative than +50 mV, state (3) with the K(+) pump "on, " and state (4) with the pump dominant, but affected by high K(+). A model for the Ventricaria membrane system is proposed.

Plasma Membrane Na+ Transport in a Salt-Tolerant Charophyte (Isotopic Fluxes, Electrophysiology, and Thermodynamics in Plants Adapted to Saltwater and Freshwater).

In salt-tolerant Chara longifolia, enhanced Na+ efflux plays an important role in maintaining low cytoplasmic Na+. When it is cultured in fresh water (FW), C. longifolia has a higher Na+ efflux than the obligate FW Chara corallina, although pH dependence and inhibitor profiles are similar for both species (J. Whittington and M.A. Bisson [1994] J Exp Bot 45: 657-665). When it is cultured in saltwater, C. longifolia has a Na+ efflux of 264 [plus or minus] 14 nmol m-2 s-1 at pH 7, 13 times higher than FW-adapted cultures and 31 times higher than C. corallina. As in FW-adapted plants, efflux is highest at pH 5, but pH dependence is less steep and more linear in cells adapted to saltwater. In plants of both species from FW cultures, Na+ efflux is inhibited by Li+ at pH 5 but not at pH 7 or 9, whereas in the salt-adapted C. longifolia, Li+ inhibits Na+ efflux at pH 7 and 9 but not at pH 5. Amiloride inhibits Na+ efflux in salt-adapted cells but not in FW cells. We conclude that a new type of Na+ efflux system is induced in salt-adapted plants, although both systems have characteristics suggestive of a Na+/H+ antiport. In all cases, a 1:1 Na+/H+ antiport would have sufficient energy to maintain the cytoplasmic Na+ activities measured at pH 5 and 7 but not at pH 9, which suggests that another efflux system must be operating at pH 9.

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities.

Plasma membranes were isolated using the aqueous polymer two-phase partition method from the algae Chara corallina and Chara longifolia, algae which differ in their ability to grow in saline environments. Enrichment of plasma membrane and depletion of tonoplast relative to the microsomal fraction was monitored using phosphohydrolase assays and cross-reactions to antibodies raised against higher plant transporters. Antibodies to the vacuolar ATPase and pyrophosphatase cross-reacted with epitopes in the microsomal fraction, but showed little affinity for the plasma membrane fraction. Pyrophosphatase activity also declined in the plasma membrane fraction relative to the microsomal fraction. The V-type H(+)-ATPase activity, sensitive to nitrate or bafilomycin, was low in both fractions, though the cross-reaction to the antibody was reduced in the plasma membrane fraction. By contrast, the antibody recognition of a P-type H(+)-ATPase amino acid sequence from Arabidopsis did not occur strongly in the anticipated 90-100 kDa range. While there was enhanced recognition of a polypeptide at around 140 kDa in the plasma membrane fraction, salt treatment of Chara longifolia resulted in plasma membrane fractions with reduced amounts of this epitope, but no change in vanadate-sensitive ATPase activity, suggesting that it does not represent the only P-type ATPase. Microsomal membranes from salt-adapted C. longifolia have higher reactivity with the antibody to the tonoplast ATPase.

Passive Proton Conductance Is the Major Reason for Membrane Depolarization and Conductance Increase in Chara buckellii in High-Salt Conditions.

Chara buckellii G.O.A., a salt-tolerant alga, has a less negative membrane potential (Em) when cultured in saline medium (artificial Waldsea water) than when cultured in freshwater. The cell hyperpolarizes and membrane conductance (Gm) decreases when the external medium is changed from Waldsea control solution (WCS), a high-salt medium, to low-salt medium containing sufficient sorbitol to generate the same osmotic potential as WCS. Banding pattern and proton flux experiments show that C. buckellii has higher passive proton influx in the alkaline band in high-salt medium than in low-salt medium. Decrease of the passive proton influx by darkness or low external pH dramatically hyperpolarizes the membrane and decreases the conductance. The pH dependence curves of Em and Gm also indicate the existence of high passive proton conductance (GH) in C. buckellii. Ion substitution experiments show that Em and Gm of saltwater cells are not dependent on K+, Na+, Cl-, or SO42+. Mg2+ also affects Em and Gm, but its effect is probably on GH. We conclude that GH is the most important cause of the membrane depolarization and conductance increase in the saltwater alga C. buckellii.

Chara plasmalemma at high pH: voltage dependence of the conductance at rest and during excitation.

The high pH state of Chara plasmalemma (Bisson, M.A., Walker, N.A. 1980. J. Membrane Biol. 56:1-7) was investigated to obtain detailed current-voltage (I/V) and conductance-voltage (G/V) characteristics in the pH range 7.5 to 12. The resting conductance started to increase at a pH as low as 8.5, doubling at pH 9.5, but the most notable increases occurred between pH 10.5 and 11.5, as observed previously (Bisson, M.A., Walker, N.A. 1980. J. Membrane Biol. 56:1-7; Bisson, M.A., Walker, N.A. 1981. J. Exp. Bot. 32:951-971). The slopes (and shapes) of the I/V curves varied even over minutes, suggesting a shifting population of open channels. Possible contributions of the permeabilities to H+ and OH-, PH and POH, respectively, to the increase in membrane conductance were calculated in the pH range 8.5 to 12. If PH is the main cause for the increase in conductance, it would have to rise by three orders of magnitude between pH 8.5 and 11.5, implying an enormous increase in the open-channel population as pH rises. On the other hand, a comparatively constant POH over that pH range would result in an increase in conductance due to the rise of OH- concentration. This indicates unchanging open-channel population. The transient excitation conductances at pH 7.5 and 11.5 were compared at a range of membrane PD (potential difference) levels. At more positive PD levels (near 0) the transient conductances showed little change as pH was increased. However, near the excitation threshold the conductance at high pH was slower to reach peak and its amplitude was diminished compared to that at neutral pH. This effect was found to be partially due to the pH change itself and partially due to less negative membrane PD at high pH. The changes in excitation transients developed gradually as pH of the medium was increased. These findings are discussed with a recent model of excitation in mind (Shiina, T., Tazawa, M. 1988. J. Membrane Biol. 106:135-139).

Chara buckellii, a Euryhaline Charophyte from an Unusual Saline Environment : III. Time Course of Turgor Regulation.

The time course of turgor regulation of the euryhaline giant-celled alga, Chara buckellii, is presented. Isolated intermodal cells were challenged by increasing or decreasing the external osmotic pressure by 150 milliosmoles per kilogram with all ions in the media or by dilution, respectively. Regulation following hypotonic stress was complete within 48 hours whereas regulation following hypertonic stress required between 96 and 144 hours. The change in internal osmotic pressure could be entirely accounted for by changes in vacuolar KCl in response to hypotonic stress, but this ion pair only accounted for 45% of the change in response to hypertonic stress. The membrane potential of C. buckellii is normally hyperpolarized with respect to the equilibrium potential for K(+) (E(K)). The membrane depolarized to a level close to E(K) in response to hypotonic treatment and this was accompanied by a transient increase in membrane conductance. In response to hypertonic stress, the membrane hyperpolarized transiently, then repolarized to a level close to the control. This was accompanied by a temporary decrease in membrane conductance. The data are discussed with respect to the ecological significance of the time course and ion transport mechanisms during turgor regulation.

Inhibitors of proton pumping: effect on passive proton transport.

Reported inhibitors of the Characean plasmalemma proton pump were tested for their ability to inhibit the passive H(+) conductance which develops in Chara corallina Klein ex Willd. at high pH. Diethylstilbestrol inhibits the proton pump and the passive H(+) conductance with about the same time course, at concentrations that have no effect on cytoplasmic streaming. N-Ethylmaleimide, a sulfhydryl reagent which is small and relatively nonpolar, also inhibits both pumping and passive conductance of H(+). However, it also inhibits cytoplasmic streaming with about the same time course, and therefore could not be considered a specific ATPase inhibitor. p-Chloromercuribenzene sulfonate (PCMBS), a sulfhydryl reagent which is large and charged and hence less able to penetrate the membrane, does not inhibit pumping or conductance at low concentration. At high concentration, PCMBS sometimes inhibits pumping without affecting H(+) conductance, but since streaming is also inhibited, the effect on the pump cannot be said to be specific. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water soluble carbodiimide, weakly inhibits both pump and conductance, apparently specifically.

Osmoregulation or turgor regulation in chara?

Chara corallina Klein ex Willd. wm. R.D.W. (= C. australis R. Br.), a fresh water alga, maintains a constant internal osmotic pressure when external osmotic pressure is increased. This results in a decrease in turgor pressure. Chara osmoregulates effectively in the presence of high CaCl(2) and raffinose, but is less efficient in response to increased NaCl. Decreasing external pH from 7 to 5 results in a decrease in turgor, but increasing it to values as high as 9 has no effect. Increasing the daily amount of light from 0.5 to 24 hours has no effect on turgor.

Calcium effects on electrogenic pump and passive permeability of the plasma membrane of Chara corallina.

Removal of Ca2+ from the medium results in depolarization of the Chara internodal cell and an increase in membrane conductance (Gm). The increase in conductance is associated with an increase in K+ conductance, as judged by Ca2+ effects on the K+ dependence of clamp current. The voltage dependence of Gm is also affected by Ca2+, as is the time course of the response of clamp current to a step change in voltage. Mg2+ restores the low conductance and the fast response to a voltage change, but not hyperpolarization at neutral pH, suggesting that there is an additional, independent effect on the electrogenic pump. The membrane does not show the normal ability to increase proton conductance at high pH in the absence of Ca2+; this is also restored by Mg2+ as well as by Ca2+.

Vacuolar and cytoplasmic pH, ion composition, and turgor pressure in Lamprothamnium as a function of external pH.

Ionic responses to alteration in external and internal pH were examined in an organism from a marine-like environment. Vacuolar pH (pH(v)) is about 4.9-5.1, constant at external pH (pH(o)) 5-8, while cytoplasmic pH (pH(c)) increases from 7.3 to 7.7. pH(c) regulation fails above pH(o) 9, and this is accompanied by failure of turgor regulation. Na(+) increases above pH(o) 9, while K(+) and Cl(-) decrease. These changes alone cannot however explain the alterations in turgor. Agents known to affect internal pH are also tested for their effect on ion relations.

Diffusion of carbon dioxide through lipid bilayer membranes: effects of carbonic anhydrase, bicarbonate, and unstirred layers.

Diffusion of (14)C-labeled CO(2) was measured through lipid bilayer membranes composed of egg lecithin and cholesterol (1:1 mol ratio) dissolved in n-decane. The results indicate that CO(2), but not HCO(3-), crosses the membrane and that different steps in the transport process are rate limiting under different conditions. In one series of experiments we studied one-way fluxes between identical solutions at constant pCO(2) but differing [HCO(3-)] and pH. In the absence of carbonic anhydrase (CA) the diffusion of CO(2) through the aqueous unstirred layers is rate limiting because the uncatalyzed hydration-dehydration of CO(2) is too slow to permit the high [HCO(3-)] to facilitate tracer diffusion through the unstirred layers. Addition of CA (ca. 1 mg/ml) to both bathing solutions causes a 10-100-fold stimulation of the CO(2) flux, which is proportional to [HCO(3-)] over the pH range 7-8. In the presence of CA the hydration- dehydration reaction is so fast that CO(2) transport across the entire system is rate limited by diffusion of HCO(3-) through unstirred layers. However, in the presence of CA when the ratio [HCO(3-) + CO(3=)]:[CO(2)] more than 1,000 (pH 9-10) the CO(2) flux reaches a maximum value. Under these conditions the diffusion of CO(2) through the membrane becomes rate limiting, which allows us to estimate a permeability coefficient of the membrane to CO(2) of 0.35 cm s(-1). In a second series of experiments we studied the effects of CA and buffer concentration on the net flux of CO(2). CA stimulates the net CO(2) flux in well buffered, but no in unbuffered, solutions. The buffer provides a proton source on the upstream side of the membrane and proton sink on the downstream side, thus allowing HCO(3-) to facilitate the net transport of CO(2) through the unstirred layers.