Synergy between Tannerella forsythia and Fusobacterium nucleatum in biofilm formation.

During dental plaque formation, the interaction of different organisms is important in the development of complex communities. Fusobacterium nucleatum is considered a 'bridge-organism' that facilitates colonization of other bacteria by coaggregation-mediated mechanisms and possibly by making the environment conducive for oxygen intolerant anaerobes. These studies were carried out to determine whether coaggregation between F. nucleatum and Tannerella forsythia is important in the formation of mixed species biofilms. Further, the role of BspA protein, a surface adhesin of T. forsythia, in coaggregation and biofilm formation was investigated. The results showed the development of synergistic mixed biofilms of F. nucleatum and T. forsythia when these bacteria were cocultured. The BspA protein was not involved in biofilm formation. Though BspA plays a role in coaggregation with F. nucleatum, presumably other adhesins are also involved. The synergistic biofilm formation between the two species was dependent on cell-cell contact and soluble components of the bacteria were not required. This study demonstrates that there is a positive synergy between F. nucleatum and T. forsythia in the development of mixed biofilms and that the cell-cell interaction is essential for this phenomenon.

Mechanically induced calcium movements in astrocytes, bovine aortic endothelial cells and C6 glioma cells.

Forces applied to resting primary astrocytes, bovine aortic endothelial cells and C6 glioma cells with collagen-coated magnetite particles produce a fast transient change of intracellular Ca(2+). It peaks in the micromolar range as measured by Fura-2. This mechanical response adapts within seconds so that repeated stimulation causes smaller responses requiring >10 min for recovery. When cytoplasmic Ca(2+) is high after treating with ATP, cyclopiazonic acid and thapsigargin, stimulation causes a transient decrease in Ca(2+). In these three cell types, no influx of ions is required for Ca(2+) elevation showing the response is not caused by activation of plasmalemmal mechanosensitive channels. Approximately half the cells tested showed similar behavior, while the other half, such as fibroblasts, required extracellular Ca(2+). The Ca(2+) response is not temperature sensitive suggesting the possible involvement of intracellular mechanosensitive channels. We tested a number of second messenger reagents and were only able to block the response in BAECs, but not C6 glioma cells, with Xestospongin C, a blocker of IP(3)-activated channels. Despite the lack of a causal involvement of plasmalemmal mechanosensitive channels, mechanical stimulation immediately activates a persistent Mn(2+) influx pathway. This Mn(2+) pathway may be mechanosensitive channels, Ca(2+)-activated cation channels or depletion-activated Ca(2+) channels.

Visual loss after spine surgery: a survey.

OBJECTIVE: To survey a large number of neurosurgical spine surgeons for data regarding the presence of risk factors in patients experiencing visual loss after spine surgery. METHODS: A survey was sent to current members (as of 1997) of the American Association of Neurological Surgeons/Congress of Neurological Surgeons, Section on Disorders of the Spine and Peripheral Nerves, with questions focusing on intraoperative factors that may predispose patients to perioperative visual loss. RESULTS: Two hundred ninety surveys were returned, and 24 patients with visual loss after spine surgery were reported by 22 surgeons. Although many of these patients had probable causative factors for visual loss after surgery (e.g., hypotension, low hematocrit level, coexisting disease), some did not (n = 8). CONCLUSION: These results suggest the necessity of a high index of suspicion for evolving perioperative visual loss even in the absence of risk factors.

Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL.

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

Storing analog data in a video record.

We have designed a simple device that will encode, in machine readable form, multiple analog data in a video record. The analog data is visible to the user within the video frame permitting visual correlation of these signals with activity observed in the image. By superimposition of both analog and video data sets, the two are tightly synchronized and remain so in all copies of the data. The analog data can be separated from the image following digitization by a frame grabber. The bandwidth for each of the five analog channels is approximately 5 kHz. The device, which is essentially an eight channel video multiplexor, includes a video channel, a field counter, an amplitude calibration signal and five analog data channels. The amplitude calibrator allows corrections for gain errors that are particularly prevalent when data is stored on video tape.

Voltage dependence of mouse acetylcholine receptor gating: different charge movements in di-, mono- and unliganded receptors.

1. The voltage dependence of binding and gating in wild-type and mutant recombinant mouse nicotinic acetylcholine receptors (AChRs) was examined at the single-channel level. 2. The closing rate constant of diliganded receptors decreased e-fold with approximately 66 mV hyperpolarization in both wild-type (adult and embryonic) and mutant receptors. The opening rate constant of a mutant receptor (alpha Y93F) was not voltage dependent. 3. The voltage dependence of closing in monoliganded receptors was examined in several receptors having a mutation in the binding site (alpha G153S) or pore region (alpha L251C and epsilon T264P). The closing rate constant of these monoliganded receptors decreased e-fold with approximately 124 mV hyperpolarization. 4. The voltage dependence of closing and opening in unliganded receptors was examined in two receptors having a mutation in the pore region (alpha L251C and epsilon T264P). Neither the closing nor the opening rate constants of unliganded receptors were voltage dependent. 5. If z if the amount of charge that moves during channel closure and delta is the distance (as a fraction of the electric field) that the charge moves, we conclude that z delta = 0.4 in diliganded receptors, 0.2 in monoliganded receptors, and 0.0 in unliganded receptors. It is likely that charges on the protein, rather than the agonist molecule, move z delta = 0.2 after each ACh molecule has bound. 6. The results suggest that unliganded openings arise from a local, concerted change in the structure of the pore (channel opening) that does not involve the net movement of charged residues. We speculate that as a consequence of agonist binding, charged moieties in the protein change their disposition so that they move with respect to the electric field when the channel gates. The results are consistent with the idea that there is semi-independent movement of distinct domains during AChR gating.

Ca2+ uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels.

Hypotonic cell swelling triggers an increase in intracellular Ca2+ concentration that is deemed responsible for the subsequent regulated volume decrease in many cells. To understand the mechanisms underlying this increase, we have studied the Ca2+ sources that contribute to hypotonic cell swelling-induced Ca2+ increase (HICI) in GH3 cells. Fura 2 fluorescence of cell populations revealed that extracellular, but not intracellular, stores of Ca2+ were required. HICI was abolished by nifedipine, a blocker of L-type Ca2+ channels, and Gd3+, a nonspecific blocker of stretch-activated channels (SACs), suggesting two components for the Ca2+ membrane pathway: L-type Ca2+ channels and SACs. Using HICI as an assay, we found that venom from the spider Grammostola spatulata could block HICI without blocking L-type Ca2+ channels. The venom did, however, block SAC activity. This suggests that Ca(2+)-permeable SACs, rather than L-type Ca2+ channels, are the sensing elements for HICI. These results support the model for volume regulation in which SACs, activated by an increase of the membrane tension during hypotonic cell swelling, trigger HICI, leading to a volume decrease.

Mechanically induced calcium mobilization in cultured endothelial cells is dependent on actin and phospholipase.

We sought to evaluate the mechanisms by which mechanical perturbation elevates intracellular calcium in endothelial cells. We report that the transient elevation in intracellular calcium in cultured bovine aortic endothelial cells (BAEC) in response to gentle perturbation with the side of a micropipette was not blocked by depolarization (external K+, 130 mmol/L), nifedipine (10 mumol/L), or Bay K 8644 R(+) (10 mumol/L). Thus, voltage-dependent calcium channels were not involved in the response. Also, amiloride (10 mumol/L) and tetraethylammonium (1 mmol/L) had no effect on calcium mobilization, indicating that Na+ and K+ transporters were not involved. Pretreatment of the cells with the phospholipase C and phospholipase A2 inhibitor manoalide (10 mumol/L) for 10 minutes at 37 degrees C completely abolished the calcium response, as did a 10-minute pretreatment with the inhibitor of actin polymerization, cytochalasin B (1 mumol/L). We observed an inhibitory effect of the phospholipase A2 and phospholipase C inhibitor 4-bromophenacyl bromide (10 mumol/L) on the mechanical response of BAEC that was not as potent as that observed with manoalide. To examine the role of arachidonic acid (AA) and subsequent metabolites that may be released after a putatively mechanical activation of phospholipase A2, we exposed BAEC to exogenous AA. We found that continued exposure of BAEC for 5 minutes to 10 nmol/L to 10 mumol/L AA caused no elevation of intracellular calcium. If mechanical stimulation activates phospholipase A2, the liberated AA and subsequent metabolites do not appear to have much effect on BAEC intracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical perturbation of cultured human endothelial cells causes rapid increases of intracellular calcium.

In first-passage human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (passages 13-16), exposure to gentle mechanical perturbation using a micropipette caused a transient rise in intracellular calcium concentration ([Ca2+]i). The increase in calcium concentration ([Ca2+]) occurred each time the cell was nudged. Three responses were evoked in each of 27 cells using 5 independent HUVEC harvests. Increase in [Ca2+] returned to near baseline levels within approximately 30 s. The stimulus did not cause membrane puncture, as indicated by 1) absence of rapid dye leakage, 2) regulated nature of the [Ca2+] response, 3) absence of membrane blebbing, and 4) repeatable nature of the response in the same cell. As an alternative stimulus, we created very narrow fluid streams (1- to 2-microns diam) from a pressurized pipette that generated shear stresses of approximately 0.001-0.1 dyn/cm2 on the cells. However, these low-shear streams had little effect on [Ca2+]i. The poke-induced change in [Ca2+] was not blocked by lowering extracellular [Ca2+] ([Ca2+]o; 10 microM). In the absence of [Ca2+]o, however, HUVEC did not respond to the first poke, indicating a requirement for some [Ca2+]o as a mediator of signaling. After several poke-induced responses, [Ca2+]i could still be released by caffeine (100 microM), indicating the integrity of the intracellular release mechanism(s). These studies indicate that the response of an endothelial cell to a membrane-deforming event involves a priming step utilizing [Ca2+]o, which facilitates the transient increase of [Ca2+]i.

Calcium imaging of mechanically induced fluxes in tissue-cultured chick heart: role of stretch-activated ion channels.

Heart rate and contractility are sensitive to stretch. To better understand the origin of these effects, we have studied the effect of mechanical stimuli on a model system of tissue-cultured heart cells. Gently prodding cells with a pipette produced a Ca2+ influx that often led to waves of calcium-induced calcium release (CICR) spreading from the site of stimulation. Ca2+ release could also be produced by pulling on neighboring cells. The response was blocked by removing extracellular Ca2+ or by adding 20 microM Gd3+ to normal saline. The mechanical sensitivity probably arose from stretch-activated ion channels (SACs) based on several lines of evidence. Chick heart cells contain nonselective cation SACs that pass Ca2+ as well as Na+ and K+. Both the SACs and the fluorescence response are blocked by 20 microM Gd3+. Removal of Ca2+ from the extracellular medium blocked the fluorescent response. Cultures without SACs (grown in the absence of embryo extract) had no mechanically induced fluxes. These data contradict the recent claim that SAC activity is a patch-clamp artifact (C.E. Morris and R. Horn, Science Wash. DC 256: 1246-1249, 1991). The SACs had a density of approximately 1/micron 2 and were expected to pass less than 20 fA of Ca2+ current under physiological conditions. The change in intracellular concentration of Ca2+ ([Ca2+]i) resulting from activation of SACs may be too small to induce CICR unless the channels pass current into a restricted space (N. LeBlanc and J.R. Hume, Science Wash. DC 248: 372, 1990).(ABSTRACT TRUNCATED AT 250 WORDS)

Stretch-activated ion channels in growth cones of snail neurons.

Using single-channel recording, we show that neurons contain ion channels sensitive to membrane tension. Neurons isolated from the snail, Lymnaea stagnalis, actively rearborized in culture yielding cell bodies and growth cones suitable for patch clamping. All neurons contained, in both their soma and growth cones (at a density of approximately 1-2 micron-2), stretch-activated channels highly selective for K+. The presence of this mechanosensitive channel in the motile region of the neuron, a region characterized by insertion of new membrane--the growth cone--is of particular interest. Under physiological conditions, the channel was permeable to K+, but not to Na+ or Cl-. Its conductance to K+ under these conditions was approximately 44 pS. Channel activation was steeply dependent on membrane tension, showing thresholds at between -50 to -100 mm Hg (suction was applied through the recording pipette). Kinetic analysis indicated that the stretch-dependent increase in the channel's open probability was related to a long closed state rather than to one of the open states. Given the importance of Ca2+ in the regulation of growth cone motility, we speculate that this stretch-activated K+ channel could play a role in neurite elongation by a tension-dependent modulation of membrane voltage which in turn would act on voltage-gated Ca2+ channels.

Stretch-inactivated ion channels coexist with stretch-activated ion channels.

Stretch-activated ion channels of animal, plant, bacterial, and fungal cells are implicated in mechanotransduction and osmoregulation. A new class of channel has now been described that is stretch-inactivated. These channels occur in neurons, where they coexist with stretch-activated channels. Both channels are potassium selective. The differing stretch sensitivities of the two channels minimize potassium conductance over an intermediate range of tension, with the consequence that, over this same range, voltage-gated calcium channels are most readily opened. Thus, by setting the relation between membrane tension and transmembrane calcium fluxes, stretch-sensitive potassium channels may participate in the control of calcium-dependent motility in differentiating, regenerating, or migrating neurons.

Activation by curare of acetylcholine receptor channels in a murine skeletal muscle cell line.

Curare action on nicotinic acetylcholine receptors has a number of facets, of which the best known is competitive antagonism. Here we describe the weak agonist action of 10(-5) M curare on the murine skeletal muscle cell line, G8. Although curare induces no depolarization in G8 cells, single-channel recordings reveal short-lived curare-induced currents. A feature of these brief events is the multiplicity of conductance levels (of the four levels with conductances of 48, 37, 14, and 6 pS, none had a lifetime greater than 1.5 ms). Most well-resolved events (about 17% of which are to a subconductance) last less than 0.5 ms, with activation occurring predominantly as isolated events rather than in bursts. Agonism is not, however, a high probability action for curare: calculations based on the frequency of events at half-saturating conditions suggest that curare-induced channel openings occur during less than 1% of acetylcholine receptor-curare binding episodes. The outcome is (a) an agonist action too feeble to perturb the membrane voltage and (b) a powerful competitive antagonist action.

A hot aldehyde-peroxide fixation method for electron microscopy of the free-living nematode Caenorhabditis elegans.

An improved method for the fixation of the third and fourth larval stages and adults of Caenorhabditis elegans has been developed. Worms are placed in a mixture of 1.5% paraformaldehyde and 1.0% glutaraldehyde at pH 7.0 and 70 C and the suspension promptly cooled in a water bath at 20 C for 1 hr. The fixed worms are then immersed in a mixture of 5% glutaraldehyde and hydrogen peroxide at 4 C for 1 hr, stained en bloc in uranyl acetate, and embedded in resin for electron microscopy. The procedure results in superior fixation, particularly of microfilaments and microtubules. The high temperature of the initial fixation straightens the worms and thus facilitates serial sectioning.