Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy.

We report on a rapid method for reagentless identification and discrimination of single bacterial cells in aqueous solutions using a combination of laser tweezers and confocal Raman spectroscopy (LTRS). The optical trapping enables capturing of individual bacteria in aqueous solution in the focus of the laser beam and levitating the captured cell well off the cover plate, thus maximizing the excitation and collection of Raman scattering from the cell and minimizing the unwanted background from the cover plate and environment. Raman spectral patterns excited by a near-infrared laser beam provide intrinsic molecular information for reagentless analysis of the optically isolated bacterium. In our experiments, six species of bacteria were used to demonstrate the capability of the confocal LTRS in the identification and discrimination between the diverse bacterial species at various growth conditions. We show that synchronized bacterial cells can be well-discriminated among the six species using principal component analyses (PCA). Unsynchronized bacterial cells that are cultured at stationary phases can also be well-discriminated by the PCA, as well as by a hierarchical cluster analysis (HCA) of their Raman spectra. We also show that unsynchronized bacteria selected from random growth phases can be classified with the help of a generalized discriminant analysis (GDA). These findings demonstrate that the LTRS may find valuable applications in rapid sensing of microbial cells in diverse aqueous media.

Effect of dicyclohexylcarbodiimide (DCCD) on transport parameters in the frog cornea epithelium.

Dicyclohexylcarbodiimide (DCCD) is a carboxyl group modifier and it is an inhibitor of various ATPases. Present experiments, using an in vitro preparation, were designed to study whether DCCD affected the transporters of the bullfrog cornea epithelium, specifically, the Na(+)/K(+) ATPase pump located in the basolateral membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (I(sc)) conditions. Addition of DCCD to a concentration of 10(-4) m to the tear solution gave a marked decrease in I(sc); a marked depolarization of the intracellular potential, V(o); and a significant decrease in the apical membrane fractional resistance, fR(o). There were small and variable although significant changes in the transepithelial conductance, g(t). The effects may be explained by a decrease in the basolateral membrane K(+) conductance, in combination with a partial inhibition of the Na(+)/K(+)-ATPase pump located in the basolateral membrane. There is also evidence for an increase in the apical membrane Cl(-) conductance.

Effect of pentachlorophenol (PCP) on frog cornea epithelium.

Pentachlorophenol (PCP) is a toxic substance that affects many tissues adversely. Present experiments, using an in vitro preparation, were designed to study whether PCP affected the electrophysiological parameters of the bullfrog cornea epithelium, specifically, the Na+/K+ ATPase pump and the K+ conductance located in the basolateral membrane and the Cl- conductance located in the apical membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (Isc) conditions. Addition of PCP to a concentration of 5 x 10-5 M to the tear solution gave a marked decrease in Isc; a marked depolarization of the intracellular potential, Vo; and minimal but significant decreases in the apical membrane fractional resistance, fRo, and in the transepithelial conductance, gt. Isc experiments in Cl--free solutions with amphotericin B in the tear solution confirm results indicating that PCP inhibits the active transepithelial transport mechanism and produces a small increase in the basolateral membrane resistance due to a decrease in the K+ conductance.

Effect of melittin on the apical membrane Na+ and Cl- conductances of frog cornea epithelium.

The transepithelial conductance increased with 10(-6) M melittin on the tear side of the frog cornea. This effect was attributed to the opening of an apical membrane Na+ conductance. However, the potency of the venom at this concentration (apical membrane fractional resistance (fRo) decreased to near zero) masked this and other effects. With 3 x 10(-7) M on the tear side, the effects were finite (fRo > 0) and reversible. With fRo > 0, the effects of melittin could be readily studied on the transport parameters of Na+, Cl-, and K+. Epithelial cells of intact bullfrog corneas were impaled with microelectrodes using an in vitro preparation. Under short-circuit current (Isc) conditions, 20 min after melittin Isc increased by 3.1 from 8.2 microA/cm2., fRo decreased by 18 from 51%; the intracellular potential, Vo, depolarized by 19.4 from -56.5 mV; and the transepithelial conductance, gv increased by 0.57 from 0.29 mS/cm2. Tenfold decreases in tear Na+ or Cl- concentrations changed the transport parameters consistent with the formation of a Na+ conductance and an increase in the apical membrane Cl- conductance by the venom. These conclusions were further supported by the minimal effect of melittin in Cl(-)-fre and, particularly, in Na(+)-free solutions. Changes in K+ concentration had no effect on transport parameters. These findings indicate that the effect of melittin at this low concentration is upon a Na+ channel protein and not due to nonspecific conductances.

Effect of melittin on electrophysiological parameters in the frog cornea epithelium.

The effect of melittin on electrophysiological parameters of the bullfrog cornea was studied using an in vitro preparation. Epithelial cells of corneas were impaled with microelectrodes. Experiments were done under short-circuit current (Isc) conditions. Melittin was added in concentrations of 10(-5)M to the stromal or 10(-6)M to the tear solution. The effects of melittin were as follows: (i) stromal side, a decrease in Isc; an increase in the apical membrane fractional resistance, fRo; no change in the transepithelial conductance, gt; and a depolarization of the intracellular potential, Vo; (ii) tear side, an initial (first 10 min) increase and then a decrease in Isc; a decrease in fRo; an initial (first 10 min) increase with subsequent small decrease in gt; and a depolarization of Vo. Changes in tear Na+, but not in tear K+, with melittin present in tear solution, induced changes in some electrical parameters. The effects on the tear side may be explained by opening of nonspecific channels in the apical membrane with some specificity for Na+ channel. The subsequent effects on the tear and the effects on the stromal side may be explained by an inhibition of the primary transport system, that is, of the Na+/K+ -ATPase pump located in the basolateral membrane. In these experiments, there was no evidence of opening of channels in the basolateral membrane.

Calcium and the effects of ultrasound on frog skin.

Therapeutic ultrasound is used to enhance the repair of soft tissue, muscle, etc., and because many of the cellular reactions involved in these processes are dependent on the intracellular availability of free calcium ions, it becomes important to study the effects of ultrasound in the presence and the absence of calcium ions. Using frog skin as a biological model, the effect of therapeutic ultrasound (300 mW/cm2 1 MHz CW) was investigated. Sonication for two minutes caused a significantly larger increase in total ionic conductance (Gt) in the presence of calcium ions (140% vs. 27%). However, the time constant for Gt to return to steady state was significantly longer in calcium-free solutions (122 vs. 18 min.). This study demonstrates that the biological effects of ultrasound are influenced by calcium ions. Furthermore, the recovery time constants confirm recent findings regarding the function of calcium ions in the formation of tight junctions. The role of free radicals produced by cavitation and calcium potentiated lipid and protein peroxidation is discussed.

Inactivation of firefly luciferase and rat erythrocyte ATPase by ultrasound.

Previous work in our laboratories has shown that, amongst other effects, irradiation of frog skin with low intensity ultrasound causes reductions in the chemical driving force of the short-circuit current. This indicated that either the Na/K dependent ATPase or ATP availability were being reduced. We measured the effect of ultrasound irradiation on ATP and NA/K-dependent ATPase from inverted erythrocyte ghosts and on firefly luciferin and luciferase activity. Our findings demonstrate that ultrasonic cavitation-induced sonochemical reactions were responsible for irreversible inactivation of luciferase and ATPase but had little or no effect on ATP and luciferin. We measured the levels of hydrogen peroxide generated by ultrasound under the conditions of our experiments and found that it could account for only part of the enzyme inactivation observed. Free radical scavengers/antioxidants were capable of fully protecting the enzymes from ultrasound-induced inactivation. These findings demonstrate that, in addition to hydrogen peroxide, free radicals generated by ultrasound are responsible for the effects.

Acoustic cavitation produced by microsecond pulses of ultrasound: a discussion of some selected results.

Because of its extensive utilization in clinical practice, and because the subjects examined are often fragile and sensitive to trauma, the safety of diagnostic ultrasound has always been of concern. Of the various mechanisms through which ultrasound could act in a manner deleterious to a patient, acoustic cavitation, should it occur, appears to possess significant potential for biological damage. This paper reviews several recent reports of progress by our two groups and demonstrates the conditions under which cavitation has been observed by microsecond pulses of ultrasound. Although these results give no indications that diagnostic ultrasound may pose a true risk to a patient, they do indicate that in vivo cavitation may occur under certain conditions.

Effect of amphotericin B and Cl- removal on basolateral membrane K+ conductance in frog corneal epithelium.

Increase in stromal K+ concentration from 4 to 79 mM in an in vitro preparation of the frog cornea, in Cl(-)-free solutions, did not change the apical membrane fractional resistance, fR0, or the transepithelial conductance, gt; it depolarized the intracellular potential, V0, by 38 mV and decreased the short-circuit current, Isc by 2.9 microA/cm2. These changes were similar to those observed for the same increase in stromal K+ in control solutions except for the increase in gt in the latter. When stromal K+ was increased with 10(-5) M amphotericin B, AmB, in the tear solution, fR0 increased by 0.27 in control solutions and by 0.08 in Cl(-)-free solutions; respectively, gt increased by 0.40 and by 0.17 mS/cm2; Isc decreased by 12 and by 11 mS/cm2; V0 depolarized by 9 and by 9.5 mV. These results support the concept that: (i) entrance of Cl- into the cell is responsible in part for the bioelectrical changes observed when stromal K+ is increased; and (ii) AmB decreases the partial K+ conductance in the basolateral membrane of the frog cornea epithelium by a decrease in intracellular K+.

The significance of membrane changes in the safe and effective use of therapeutic and diagnostic ultrasound.

The cellular changes, such as alterations in motility and the stimulation of synthesis and secretion, induced by relatively low intensities of therapeutic ultrasound (e.g. 500 mW cm-2, SAPA; 100 mW cm-2 SATA) are primarily non-thermal in origin. They appear to be associated with changes in the permeability of the cell (plasma) membrane and in the transport of ions and molecules across it, effects which have been demonstrated in cells irradiated in suspension. In epithelial tissues, both in vitro and in vivo, it has been demonstrated that not only the cellular membrane transport pathways but also the paracellular or intercellular pathways are affected. Although membrane-mediated effects can be of value therapeutically, they could produce adverse effects if they were to occur during development, for the reception and transmission by the membrane of environmental signals are involved in determination of the fate of each cell. Determination is followed by selective gene expression and differentiation, that is, by the progressive increase in structural complexity brought about by the acquisition of specialised characteristics by various cell groups. Most cells of early embryos are ionically coupled via gap junctions which provide an intercellular pathway for electrochemical signalling and the maintenance of the concentration gradients which provide the cells with positional information. Differentiation of the cells varies according to their location with respect to these gradients. Increase in the intracellular concentration of calcium ions, which has been shown to occur after exposure to therapeutic levels of ultrasound, can decrease the permeability of gap junctions and uncouple cells, in the manner which occurs when they differentiate. Ultrasonically induced increases in calcium ion concentration are thus of considerable clinical significance, since they could affect differentiation and consequently histogenesis. Modification of plasma membrane permeability and transport properties, resulting in changes in the availability and activity of second messengers such as free calcium ions, can have profound effects on cell behaviour. Calcium channels appear to be the first channels to develop in the cell membranes of embryos, and internal calcium ion concentration is known to affect the synthesis of fetal proteins. Although generally reversible at intensities of less than 500 mW cm-2, changes in membrane permeability, particularly to calcium ions, could, if prolonged, have undesirable side effects not only on embryogenesis but on late prenatal and postnatal development. It is therefore recommended that the environmental conditions, thresholds, and mechanisms involved in the production of such changes be determined, so that they can be avoided when ultrasound is used diagnostically on sensitive targets such as embryos and fetuses.

Microelectrode studies of amphotericin B on Na+ and K+ conductance in bullfrog cornea.

Addition of 10(-5) M amphotericin B to the tear solution of an in vitro preparation of the frog cornea increased the transepithelial conductance, gt, and decreased the apical membrane fractional resistance, f(R0), in the presence or absence of tear Na+ and Cl-. In the presence of tear Na+ and Cl-, amphotericin B increased the short-circuit current, Isc, from 3.9 to 8.8 microA.cm-2 and changed the intracellular potential, V0, from -48.5 to -17.9 mV probably due to a higher increase in the Na+ than in the K+ conductance. In the absence of tear Na+ and Cl-, amphotericin B decreased Isc from 5.5 to about 0 microA.cm-2 due to K+ (and possibly Na+) flux from cell to tear and changed V0 from -35.4 to -63.6 mV due to the increase in conductance of both ions. Increase in the tear K+ from 4 to 79 mM (in exchange for choline), in the presence of amphotericin B and absence of tear Na+ and Cl-, decreased f(R0) from 0.09 to 0.06, increased gt from 0.23 to 0.31 mS, increased Isc from 0.63 to 7.3 microA.cm-2, and changed V0 from -65.5 to -17.3 mV due to the change in EK in the presence of a high conductance in the tear membrane. Similar effects were observed with an increase of tear Na+. Results support the concept that the Na+ conductance opened by amphotericin B in the apical membrane is greater than the K+ conductance. Previously observed transepithelial effects of the ionophore may be explained mostly on the basis of its effect on the apical membrane.

The effect of therapeutic ultrasound on electrophysiological parameters of frog skin.

There are two groups of mechanisms through which ultrasound can affect biological systems, those of thermal origin and others of nonthermal origin. Since in almost every therapeutic application of ultrasound, movement of ions across cellular membranes is involved, it becomes important to study the effect of ultrasound on active and passive ionic conductance. In order to differentiate between thermal and nonthermal effects, a study was conducted on model systems in which the effect of temperature is known. The well-known sodium transporting epithelium, the epidermis of abdominal frog skin, was investigated and the effect of therapeutic ultrasound on its electrophysiological properties was determined. It was found that under open circuit conditions, irradiation of the skin with 1 MHz cw (60-480 mW/cm2) ultrasound caused a significant decrease (5-50%, depending on the applied power) in the transepithelial potential and resistance at room temperature (20-22 degrees C). Under short circuit conditions, also at room temperature, there was an increase in total ionic conductance (20-250%, depending on the applied power) and a decrease in the net actively transported current, measured as the short circuit current. These effects are reversible within the range of powers used. Furthermore, it was found that the magnitude of the observed changes was strongly dependent on the perfusion rate and the gas content of the bathing medium. The effect of ultrasound diminished in the presence of CO2 and was enhanced with faster perfusion rates. Pulsed ultrasound delivered at the same energy (Isata) as that of cw caused a significantly larger effect. At lower temperatures (12-14 degrees C) the effect of ultrasound was reduced. Analysis of the data reveals that the effects of ultrasound on ion transport reported here are not primarily of thermal origin but are probably due to cavitation and related effects, such as microsteaming.

Potential difference responses to nutrient K+, Cl- and Na+ changes in secreting and resting states of frog stomach.

The effects of changes in nutrient concentrations of K+, Cl- and Na+ on the transmucosal potential difference (PD) and the resistance were compared for secreting fundus and resting fundus of Rana pipiens. Increase of K+ from 4 to 40 mM, decrease of Cl- from 81 to 8.1 mM and decrease of Na+ from 102 to 10 mM gave, 10 min after the change in the secreting fundus, delta PD values of -28.2, -19.8 and -7.5 mV, respectively. In the resting fundus with SCN- inhibition, the same changes in nutrient ion concentration gave delta PD values of -20.1, -17.0 and -10.2 mV, respectively. Changes in Na+ concentration were considered in a set of experiments of high acid secreting stomachs (4 to 6 mu equiv. . h-1 . cm-2). Here, delta PD gave for 10-fold decreases in Na+ concentration in secreting fundus -4.8 mV and in resting fundus with SCN- inhibition -22.6 mV. Omeprazole inhibition gave results quite similar to those with SCN- inhibition. From these results in going from secretion to inhibition, it follows that the increment of K+ conductance if it increased was lower than the increase in NaCl symport conductance since the change in delta PD for K+ decreased and that for Na+ increased. Also HCO3- conductance increased with inhibition. After SCN- inhibition the transmucosal resistance initially increased and later decreased. The decrease can be accounted for by the increase in conductance of the NaCl symport pathway and of the HCO3- pathway.

Temperature dependence of transcellular and intracellular parameters of frog skin.

Studies on the mechanism(s) for temperature dependence of sodium transport in tight epithelia reported in literature, have primarily been derived from transcellular studies. No attempts were made to discriminate between trans and paracellular conduction pathways nor is it possible from such measurements to examine the contribution of the apical and/or basolateral cellular membranes to the overall temperature dependency of the transport process. In this study we report on the temperature dependence of intracellular as well as transcellular parameters. Using a specific protocol, which provides the temperature coefficients for the amiloride displaceable sodium current, cellular conductance and the apical and basolateral membrane, conductances. Temperature coefficients derived from the slopes of Arrhenius plots of these parameters were found to be very similar (9-10 KCal/mole). The intracellular potential before and after amiloride showed weak temperature dependence (less than 1 mV/degrees C), however. On the basis of our results here, and in view of certain theoretical considerations, we conclude that the inhibition of Na+ transport across frog skin by decreasing the temperature is primarily due to a decrease in the permeability of the apical and basolateral membranes. This decrease in permeability is brought about by changes in the viscosity of water and the radius of the moving particle; in this case sodium plus hydration shell. The effect of temperature on the intracellular potential could be partially accounted for by changes in the equilibrium-potential for potassium and in part to changes in the rheogenic transport component for sodium across the basolateral membrane.

Inhibition of acid secretion of fundus of Rana pipiens with a high concentration of potassium on the secretory side.

Inhibition of acid secretion of the frog fundus is generally accompanied by an increase in transmucosal resistance, Rt, and in potential difference, PD (nutrient normally positive). These results are predicted for the intact tissue by an electrogenic proton pump. It has been suggested that the increase in PD with inhibition can also be explained by a neutral proton pump. The latter model postulates a K+ diffusion potential across the secretory (lumen-facing) membrane tending to make the secretory side positive. Upon inhibition, the [K+] in the lumen is assumed to increase, which decreases the diffusion potential, resulting in an increase in the positivity of the nutrient side. To test this theory, we determined the effects of inhibition with a high [K+] on the secretory side. With a high [K+] in the lumina, inhibition would result in only a small change in the ratio of K+ in the cell to that in the lumina, and hence a small change in the diffusion potential. We found, however, that inhibition increased the PD essentially the same as in the controls. With inhibition the resistance also increased with high secretory K+. Elevating the secretory K+ during secretion produced a 44% decrease in Rt indicating a large increase in luminal K+. We conclude that the results are not compatible with the K+ diffusion potential model but are those predicted by the electrogenic concept.

Potential difference responses to secretory K+, Na+ and HCO3- changes in secreting and resting states of frog stomach in Cl(-)-free media.

The effects of changes in secretory concentrations of K+, Na+ and HCO3- on transmucosal potential difference (PD) and resistance in Cl(-)-free (SO4(2-)) solutions were compared for secreting fundus and resting fundus of Rana pipiens. In the resting fundus experiments, histamine was not present in the nutrient solution and cimetidine was primarily used to obtain acid inhibition. Increase of K+ from 4 to 80 mM, decrease of Na+ from 156 to 15.6 mM and decrease of HCO3- from 25 to 5 mM gave, 10 min after the change, in the secreting fundus delta PD values of 39.7, -11.9 and 3.2 mV, respectively. In the resting fundus, 1.5 to 2 h after the addition of cimetidine, the same changes in secretory ion concentration gave delta PD values of 12.2, -5.6 and 1.5 mV, respectively. Replacement of cimetidine with SCN and without histamine yielded a delta PD somewhat lower than that in cimetidine, namely 9 mV for a K+ change from 4 to 80 mM. Subsequent addition of histamine with SCN present gave a delta PD of about 21 mV. The change in PD was attributed to histamine increasing the secretory membrane area, leading to an increase in K+ conductance. Another possibility is that histamine increases the K+ conductance per se.