Large Transient Increase Research Articles

Dynamics of pivoted slider bearings

We obtain the full equations of motion for a wide, pivoted, slider bearing. These are used to review the choice of the optimal position for the pivot point, to discuss its response to time-dependent sliding velocity, and to determine the stability of the motion. The case of an abrupt acceleration of the slider, which results in a large transient increase in the resistive force, is surprisingly complicated. We also discuss a general transversion formula for changing the dependent variables in Stokes flow problems.

Electrophysiologic and Morphologic Strain Differences in a Low-Dose NaIO3-Induced Retinal Pigment Epithelium Damage Model.

PurposeWe aimed to explore differences in the NaIO3-elicited responses of retinal pigment epithelium (RPE) and other retinal cells associated with mouse strains and dosing regimens.MethodsOne dose of NaIO3 at 10 or 15 mg/kg was given intravenously to adult male C57BL/6J and 129/SV-E mice. Control animals were injected with PBS. Morphologic and functional changes were characterized by spectral domain optical coherence tomography, electroretinography, histologic, and immunofluorescence techniques.ResultsInjection with 10 mg/kg of NaIO3 did not cause consistent RPE or retinal changes in either strain. Administration of 15 mg/kg of NaIO3 initially induced a large transient increase in scotopic electroretinography a-, b-, and c-wave amplitudes within 12 hours of injection, followed by progressive structural and functional degradation at 3 days after injection in C57BL/6J mice and at 1 week after injection in 129/SV-E mice. RPE cell loss occurred in a large posterior-central lesion with a ring-like transition zone of abnormally shaped cells starting 12 hours after NaIO3 treatment.ConclusionsNaIO3 effects depended on the timing, dosage, and mouse strain. The RPE in the periphery was spared from damage compared with the central RPE. The large transient increase in the electroretinography was remarkable.Translational RelevanceThis study is a phase T1 translational research study focusing on the development and validation of a mouse model of RPE damage. It provides a detailed foundation for future research, informing choices of mouse strain, dosage, and time points to establish NaIO3-induced RPE damage.

Endophilin A1 drives acute structural plasticity of dendritic spines in response to Ca2+/calmodulin.

Induction of long-term potentiation (LTP) in excitatory neurons triggers a large transient increase in the volume of dendritic spines followed by decays to sustained size expansion, a process termed structural LTP (sLTP) that contributes to the cellular basis of learning and memory. Although mechanisms regulating the early and sustained phases of sLTP have been studied intensively, how the acute spine enlargement immediately after LTP stimulation is achieved remains elusive. Here, we report that endophilin A1 orchestrates membrane dynamics with actin polymerization to initiate spine enlargement in NMDAR-mediated LTP. Upon LTP induction, Ca2+/calmodulin enhances binding of endophilin A1 to both membrane and p140Cap, a cytoskeletal regulator. Consequently, endophilin A1 rapidly localizes to the plasma membrane and recruits p140Cap to promote local actin polymerization, leading to spine head expansion. Moreover, its molecular functions in activity-induced rapid spine growth are required for LTP and long-term memory. Thus, endophilin A1 serves as a calmodulin effector to drive acute structural plasticity necessary for learning and memory.

Origins of radiation-induced attenuation in pure-silica-core and Ge-doped optical fibers under pulsed x-ray irradiation

We investigated the nature, optical properties, and decay kinetics of point defects causing large transient attenuation increase observed in silica-based optical fibers exposed to short duration and high-dose rate x-ray pulses. The transient radiation-induced attenuation (RIA) spectra of pure-silica-core (PSC), Ge-doped, F-doped, and Ge + F-doped optical fibers (OFs) were acquired after the ionizing pulse in the spectral range of [∼0.8–∼3.2] eV (∼1500–∼380 nm), from a few ms to several minutes after the pulse, at both room temperature (RT) and liquid nitrogen temperature (LNT). Comparing the fiber behavior at both temperatures better highlights the thermally unstable point defects contribution to the RIA. The transient RIA origin and decay kinetics are discussed on the basis of already-known defects absorbing in the investigated spectral range. These measurements reveal the importance of intrinsic metastable defects such as self-trapped holes (STHs), not only for PSC and F-doped fibers but also for germanosilicate optical fibers as clearly evidenced by our LNT measurements. Furthermore, our results show that fluorine co-doping seems to decrease the RIA related to the strain-assisted STHs absorption bands in both types of optical fibers. Regarding Ge-doped glasses, besides a description of the defects responsible of the RIA, highlighting the STHs' role in their transient response, we provide a clear correlation between the GeX and GeY centers’ kinetics. In conclusion, the presented results improve our understanding of the transient RIA origin in the ultraviolet and visible domains. The lack of knowledge about the defects causing the RIA in the near-infrared domain will require future studies.

Effects of Clonidine on the Cardiovascular, Renal, and Inflammatory Responses to Experimental Bacteremia.

Supra-clinical doses of clonidine appear beneficial in experimental sepsis, but there is limited understanding of the effects of clonidine at clinically relevant doses. In conscious sheep, with implanted renal and pulmonary artery flow probes, sepsis was induced by infusion of live Escherichia coli. At 24 h, a high clinical dose of clonidine (HCDC) [1.0 μg/kg/h], a low clinical dose of clonidine (LCDC) [0.25 μg/kg/h] or vehicle, was infused for 8 h. Animals developed hyperdynamic, hypotensive sepsis with acute kidney injury. The HCDC decreased heart rate (153 ± 6 to 119 ± 7 bpm) and cardiac output (5.6 ± 0.4 to 5.0 ± 0.4 L/min), with no reduction in mean arterial pressure (MAP). In contrast, LCDC increased cardiac output with peripheral vasodilatation. Both doses induced a large transient increase in urine output, an increase in plasma osmolality and, with the high dose, an increase in plasma arginine vasopressin. Sepsis increased plasma interleukin-6 (IL-6) and IL-10 and clonidine further increased IL-10 (1.6 ± 0.1 to 3.3 ± 0.7 ng/mL), but not IL-6. Clonidine reduced rectal temperature. During recovery from sepsis, MAP returned to baseline values more rapidly in the HCDC group (P < 0.001). In hyperdynamic, hypotensive sepsis, the effects of clonidine at clinically relevant doses are complex and dose dependent. HCDC attenuated sepsis-related increases in heart rate and cardiac output, with little effect on arterial pressure. It also induced a water diuresis, increased AVP, reduced body temperature, and had an anti-inflammatory action. Low-dose clonidine had similar but less pronounced effects, except that it induced moderate vasodilatation and increased cardiac output.

ATP triggers a robust intracellular [Ca2+ ]-mediated signalling pathway in human synovial fibroblasts.

What is the central question of this study? What are the main [Ca2+ ]i signalling pathways activated by ATP in human synovial fibroblasts? What is the main finding and its importance? In human synovial fibroblasts ATP acts through a linked G-protein (Gq ) and phospholipase C signalling mechanism to produce IP3 , which then markedly enhances release of Ca2+ from the endoplasmic reticulum. These results provide new information for the detection of early pathophysiology of arthritis. In human articular joints, synovial fibroblasts (HSFs) have essential physiological functions that include synthesis and secretion of components of the extracellular matrix and essential articular joint lubricants, as well as release of paracrine substances such as ATP. Although the molecular and cellular processes that lead to a rheumatoid arthritis (RA) phenotype are not fully understood, HSF cells exhibit significant changes during this disease progression. The effects of ATP on HSFs were studied by monitoring changes in intracellular Ca2+ ([Ca2+ ]i ), and measuring electrophysiological properties. ATP application to HSF cell populations that had been enzymatically released from 2-D cell culture revealed that ATP (10-100μm), or its analogues UTP or ADP, consistently produced a large transient increase in [Ca2+ ]i . These changes (i) were initiated by activation of the P2 Y purinergic receptor family, (ii) required Gq -mediated signal transduction, (iii) did not involve a transmembrane Ca2+ influx, but instead (iv) arose almost entirely from activation of endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (IP3 ) receptors that triggered Ca2+ release from the ER. Corresponding single cell electrophysiological studies revealed that these ATP effects (i) were insensitive to [Ca2+ ]o removal, (ii) involved an IP3 -mediated intracellular Ca2+ release process, and (iii) strongly turned on Ca2+ -activated K+ current(s) that significantly hyperpolarized these cells. Application of histamine produced very similar effects in these HSF cells. Since ATP is a known paracrine agonist and histamine is released early in the inflammatory response, these findings may contribute to identification of early steps/defects in the initiation and progression of RA.

Transient radon signals driven by fluid pressure pulse, micro-crack closure, and failure during granite deformation experiments

In seismically active fault zones, various crustal fluids including gases are released at the surface. Radon-222, a radioactive gas naturally produced in rocks, is used in volcanic and tectonic contexts to illuminate crustal deformation or earthquake mechanisms. At some locations, intriguing radon signals have been recorded before, during, or after tectonic events, but such observations remain controversial, mainly because physical characterization of potential radon anomalies from the upper crust is lacking.Here we conducted several month-long deformation experiments under controlled dry upper crustal conditions with a triaxial cell to continuously monitor radon emission from crustal rocks affected by three main effects: a fluid pressure pulse, micro-crack closure, and differential stress increase to macroscopic failure. We found that these effects are systematically associated with a variety of radon signals that can be explained using a first-order advective model of radon transport. First, connection to a source of deep fluid pressure (a fluid pressure pulse) is associated with a large transient radon emission increase (factor of 3–7) compared with the background level. We reason that peak amplitude is governed by the accumulation time and the radon source term, and that peak duration is controlled by radioactive decay, permeability, and advective losses of radon. Second, increasing isostatic compression is first accompanied by an increase in radon emission followed by a decrease beyond a critical pressure representing the depth below which crack closure hampers radon emission (150–250 MPa, ca. 5.5–9.5 km depth in our experiments). Third, the increase of differential stress, and associated shear and volumetric deformation, systematically triggers significant radon peaks (ca. 25–350% above background level) before macroscopic failure, by connecting isolated cracks, which dramatically enhances permeability.The detection of transient radon signals before rupture indicates that connection of initially isolated cracks in crustal rocks may occur before rupture and potentially lead to radon transients measurable at the surface in tectonically active regions. This study offers thus an experimental and physical basis for understanding predicted or reported radon anomalies.

Evolution of phenotypic plasticity and environmental tolerance of a labile quantitative character in a fluctuating environment.

Quantitative genetic models of evolution of phenotypic plasticity are used to derive environmental tolerance curves for a population in a changing environment, providing a theoretical foundation for integrating physiological and community ecology with evolutionary genetics of plasticity and norms of reaction. Plasticity is modelled for a labile quantitative character undergoing continuous reversible development and selection in a fluctuating environment. If there is no cost of plasticity, a labile character evolves expected plasticity equalling the slope of the optimal phenotype as a function of the environment. This contrasts with previous theory for plasticity influenced by the environment at a critical stage of early development determining a constant adult phenotype on which selection acts, for which the expected plasticity is reduced by the environmental predictability over the discrete time lag between development and selection. With a cost of plasticity in a labile character, the expected plasticity depends on the cost and on the environmental variance and predictability averaged over the continuous developmental time lag. Environmental tolerance curves derived from this model confirm traditional assumptions in physiological ecology and provide new insights. Tolerance curve width increases with larger environmental variance, but can only evolve within a limited range. The strength of the trade-off between tolerance curve height and width depends on the cost of plasticity. Asymmetric tolerance curves caused by male sterility at high temperature are illustrated. A simple condition is given for a large transient increase in plasticity and tolerance curve width following a sudden change in average environment.

Post‐depositional processes: What really happens to new atmospheric iron in the ocean's surface?

AbstractAbiotic iron removal processes such as scavenging can significantly and rapidly modify iron distribution in the dissolved‐colloidal‐particulate continuum. Therefore, these processes could be considered, in addition to ligand complexation, as a major control on atmospheric iron dissolution in seawater. In this work, we investigated the seasonal abiotic processes occurring once dust deposited on surface seawater using a series of artificial seeding experiments (allowing us to take into consideration the settling of particles on a 1 m depth layer). Here, we demonstrate that atmospheric dissolved iron concentration ([DFe]) is driven by the processes governed by the dissolved organic matter (DOM) pool. Following artificial dust seeding, an order magnitude range increase in the [DFe] (12 – 181 nmol L−1) was observed depending on the season. Under high and fresh DOM conditions (spring and summer), the rapid formation of aggregates induced a negative feedback on the [DFe] through scavenging, while a fraction of the DFe was likely organically complexed. In contrast, in low‐DOM surface waters (winter), aggregation was not observed, allowing a very large transient increase in [DFe] (181 nmol L−1) before being removed by adsorption onto settling particles. A key result of the findings is that depending on the age and quantity of DOM, the “lithogenic carbon pump” is likely a major pathway for organic carbon export. Modeling studies should therefore relate both atmospheric iron dissolution in seawater and the intensity of the subsequent biological response, to the age and quantity of DOM.

Extrasynaptic glutamate and inhibitory neurotransmission modulate ganglion cell participation during glutamatergic retinal waves

During the first 2 wk of mouse postnatal development, transient retinal circuits give rise to the spontaneous initiation and lateral propagation of depolarizations across the ganglion cell layer (GCL). Glutamatergic retinal waves occur during the second postnatal week, when GCL depolarizations are mediated by ionotropic glutamate receptors. Bipolar cells are the primary source of glutamate in the inner retina, indicating that the propagation of waves depends on their activation. Using the fluorescence resonance energy transfer-based optical sensor of glutamate FLII81E-1μ, we found that retinal waves are accompanied by a large transient increase in extrasynaptic glutamate throughout the inner plexiform layer. Using two-photon Ca(2+) imaging to record spontaneous Ca(2+) transients in large populations of cells, we found that despite this spatially diffuse source of depolarization, only a subset of neurons in the GCL and inner nuclear layer (INL) are robustly depolarized during retinal waves. Application of the glutamate transporter blocker dl-threo-β-benzyloxyaspartate (25 μM) led to a significant increase in cell participation in both layers, indicating that the concentration of extrasynaptic glutamate affects cell participation in both the INL and GCL. In contrast, blocking inhibitory transmission with the GABAA receptor antagonist gabazine and the glycine receptor antagonist strychnine increased cell participation in the GCL without significantly affecting the INL. These data indicate that during development, glutamate spillover provides a spatially diffuse source of depolarization, but that inhibitory circuits dictate which neurons within the GCL participate in retinal waves.

Letter to the Editor

Palmer et al. (2011) have made a detailed study of the purinergic regulation of K+ and Na+ transport by immortalized human mammary epithelial cell monolayers and provide evidence for differential effects of agonists at the apical and basolateral membranes. The authors claim that their cell culture system is a model for mammary ductal epithelial cells. Furthermore, they suggest that the results of the study help explain how mammary ductal epithelial cells modulate the ion content of milk. There are several points which need to be addressed before these conclusions can be generally accepted. Firstly, Palmer et al. (2011) claim that the mammary cells used in their study are ductal in origin. However, they provide no firm evidence to support this assumption, thus there is a possibility that the cells may indeed be alveolar. Thus, Palmer et al. (2011) need to establish whether or not the cells synthesize and secrete caseins and/or lactose when cultured in the presence of lactogenic hormones. Secondly, the authors show that purinergic agonists increase cytosolic Ca2+ which in turn stimulates Ca2+-activated K+ channels. In contrast, it has been reported that ATP has no effect on cytosolic Ca2+ in acini freshly isolated from the lactating mouse mammary gland (Sudlow & Burgoyne, 1997). In this connection, Katoh et al. (2001) have shown that ATP causes a large transient increase in cytosolic Ca2+ in bovine mammary epithelial cells; however, the effect of ATP is almost abolished by culturing cells in the presence of lactogenic hormones. In view of this, it is incumbent upon Palmer et al. (2011) to repeat their study with cells cultured in the presence of a precisely defined cocktail of lactogenic hormones such as dexamethasone, insulin and prolactin. Thirdly, Palmer et al. (2011) fail to take cognisance of a long-standing model of milk ion secretion initially proposed by Linzell & Peaker (1971a). The most salient feature of the model is that the primary secretion is not markedly modified by the ductal epithelial cells. Indeed, Linzell & Peaker (1971b) reported that the apical pole of goat mammary ductal epithelial cells in vivo is relatively impermeable to both Na+ and K+. Palmer et al. (2011) need to reconcile their data with the more physiologically relevant findings of Linzell & Peaker (1971b). Fourthly, the major osmolyte in milk is lactose, therefore experimentalists wishing to use cultured monolayers as a model to study the function of mammary cells (whether alveolar or ductal cells) should at least consider bathing the apical aspect of the epithelial cells with a solution containing lactose at a concentration similar to that found in milk. Such an approach will make the results more physiologically relevant. In this regard, Quesnell et al. (2007) have shown that the barrier properties of cultured bovine epithelial cell monolayers are governed by the electrolyte content of the solution bathing the apical aspect. Thus, monolayers whose apical aspect is exposed to a low-ionic strength medium, similar to that of milk, develop a greater trans-epithelial electrical resistance. It is imperative that caution must be exercised when interpreting the findings made with cultured cells in relation to the function of a highly regulated and complex epithelium such as the lactating mammary gland.

Monitoring The Activation Of Rhodopsin By The Transient Fluorescence Of Fluorescently Labeled Helix 8

The transient changes of the fluorescence of bovine rhodopsin in ROS membranes selectively labeled with Alexa594 at cysteine 316 in helix 8 were followed in time from 1 μs to 10 s after flash excitation of the photoreceptor. A large light-induced transient fluorescence increase was observed with time constants in the ms- range at pH6. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state MII (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6), the sequence of events is: Schiff base deprotonation, structural change, proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.

Monitoring the Conformational Changes of Photoactivated Rhodopsin from Μicroseconds to Seconds by Transient Fluorescence Spectroscopy

The transient changes of the tryptophan fluorescence of bovine rhodopsin in ROS membranes were followed in time from 1 micros to 10 s after flash excitation of the photoreceptor. Up to about 100 micros the fluorescence did not change, suggesting that the tryptophan lifetimes in rhodopsin and the M(I) intermediate are similar. The fluorescence then decreases on the millisecond time scale with kinetics that match the rise of the M(II) state as measured on the same sample by the transient absorption increase at 360 nm. Both the sign and kinetics of the fluorescence change strongly suggest that it is due to an increase in energy transfer to the retinylidene chromophore caused by the increased spectral overlap in M(II). Calculation of the Forster radius of each tryptophan from the high-resolution crystal structure suggests that W265 and W126 are already completely quenched in the dark, whereas W161, W175, and W35 are located at distances from the retinal chromophore that are comparable to their Forster radii. The fluorescence from these residues is thus sensitive to an increase in energy transfer in M(II). Similar results were obtained at other temperatures and with monomeric rhodopsin in dodecyl maltoside micelles. A large light-induced transient fluorescence increase was observed with ROS membranes that were selectively labeled with Alexa594 at cysteine 316 in helix 8. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state M(II) (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6) the sequence of events is Schiff base deprotonation, structural change, and proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.

Gill membrane remodeling with soft-water acclimation in zebrafish (Danio rerio)

Little is known regarding the ionoregulatory abilities of zebrafish exposed to soft water despite the popularity of this model organism for physiology and aquatic toxicology. We examined genomic and nongenomic changes to gills of zebrafish as they were progressively acclimated from moderately hard freshwater to typical soft water over 7 days and held in soft water for another 7 days. Gills were sampled daily and mRNA expression levels of gill Na(+)-K(+)-ATPase (NKA) alpha1a subunit, epithelium calcium channel (ECaC), carbonic anhydrase-1 and 2 (CA-1, CA-2), Na(+)/H(+) exchanger (NHE-2), V-type proton (H(+))-ATPase, and copper transport protein (CTR-1) were quantified by real-time PCR. Changes in enzyme activities of gill NKA were determined and protein levels of NKA and ECaC were quantified by Western blotting. Levels of mRNA for ECaC increased fourfold after day 6, with an associated increase in ECaC protein levels after 1 wk in soft water. CA-1 and CA-2 exhibited a 1.5- and 6-fold increase in gene expression on days 6 and 5, respectively. Likewise, there was a fivefold increase in NHE-2 expression after day 6. Surprisingly, CTR-1 mRNA showed a large transient increase (over threefold) on day 6, while H(+)-ATPase mRNA did not change. These data demonstrate a high degree of phenotypic plasticity in zebrafish gills exposed to an ion-poor environment. This not only enhances our understanding of ionoregulatory processes in fish but also highlights the need for proper experimental design for studies involving preacclimation to soft water (e.g., metal toxicity).

The laterodorsal tegmentum is essential for burst firing of ventral tegmental area dopamine neurons.

In response to behaviorally salient stimuli, dopamine (DA) neurons fire in bursts. Burst firing induces a large transient increase in synaptic DA and is regarded as the functionally relevant mode of transmission that signals reward and modulates goal-directed behavior. DA neuron burst firing is dynamically regulated by afferent inputs, and it is not present in vitro because of severing of afferent processes. However, what afferents are requisite for burst firing in vivo is not known. Here, we show that tonic input from the laterodorsal tegmental nucleus (LDTg) is required for glutamate-elicited burst firing in ventral tegmental area DA neurons of anesthetized rats. Also, after LDTg inactivation, DA neurons fire as they do in vitro (i.e., as pacemakers); even direct glutamate application fails to cause them to burst fire under these conditions. These data show that the LDTg is critical to normal DA function, and thus, pathology within this region may lead to aberrant DA signaling.

Population Coding by Globally Coupled Phase Oscillators

A system of globally coupled phase oscillators subject to an external input is considered as a simple model of neural circuits coding external stimulus. The information coding efficiency of the system in its asynchronous state is quantified using Fisher information. The effect of coupling and noise on the information coding efficiency in the stationary state is analyzed. The relaxation process of the system after the presentation of an external input is also studied. It is found that the information coding efficiency exhibits a large transient increase before the system relaxes to the final stationary state.

Cortical tubules of the mouse increase nitric oxide production when stressed with sodium ions

Renal cortex tubules contain eNOS that may be used to generate nitric oxide (NO) when cells are challenged. We propose sodium stress could lead to increased NO production through a calcium linked mechanism. NO was measured with microelectrodes at 400 or 600 mOsm NaCl in the tissue bath over 150–200 um slices of mouse kidney. Resting NO of cortical tubules was 350–400 nM compared to 75–120 nM for papilla tubules. 400 mOsm NaCl caused a transient increase in NO of 39% and a larger transient increase of 124% in NO during isotonic recovery. 600 mOsm NaCl caused a sustained increase in NO of 265% with a transient overshoot of 308% during isotonic recovery. Tubules of the papilla responded minimally to hypertonic NaCl. To separate the effect of hyperosmolarity from sodium ion stress, the cortical tubules were exposed to media made hypertonic to 400 and 600 mOsm with mannitol. The NO response to mannitol was about one-fourth that of NaCl hyperosmolarity. To determine the mechanism linking sodium stress to increased NO production, we investigated if intracellular sodium used in osmotic equilibration was exchanged for calcium ions using the sodium/calcium exchanger blocker KB-R7943. After blockade, the basal NO was reduced to 57% of control and the NO response to 600 mOsm NaCl was about 20% of normal. The data indicate sodium accumulation by cortical tubules can initiate a large increase in NO generation as sodium ions are exchanged for calcium ions. With 400 mOsm NaCl, the NO response is transient as the cell adapts to hyperosmolarity. As isotonicity is restored, a large transient increase in reflects removal of sodium ions used for osmotic equilibration for a transient increase in intracellular calcium that activates NO production. (Supported by NIH Grant HL-20605 and American Heart Association Grant-In-Aid)

Control of Volume and Turgor in Stomatal Guard Cells

Water loss from plants is determined by the aperture of stomatal pores in the leaf epidermis, set by the level of vacuolar accumulation of potassium salt, and hence volume and turgor, of a pair of guard cells. Regulation of ion fluxes across the tonoplast, the key to regulation of stomatal aperture, can only be studied by tracer flux measurements. There are two transport systems in the tonoplast. The first is a Ca(2+)-activated channel, inhibited by phenylarsine oxide (PAO), responsible for the release of vacuolar K(+)(Rb(+)) in response to the "drought" hormone, abscisic acid (ABA). This channel is sensitive to pressure, down-regulated at low turgor and up-regulated at high turgor, providing a system for turgor regulation. ABA induces a transient stimulation of vacuolar ion efflux, during which the flux tracks the ion content (volume, turgor), suggesting ABA reduces the set-point of a control system. The second system, which is PAO-insensitive, is responsible for an ion flux from vacuole to cytoplasm associated with inward water flow following a hypo-osmotic transfer. It is suggested that this involves an aquaporin as sensor, and perhaps also as responder; deformation of the aquaporin may render it ion-permeable, or, alternatively, the deformed aquaporin may signal to an associated ion channel, activating it. Treatment with inhibitors of aquaporins, HgCl(2) or silver sulfadiazine, produces a large transient increase in ion release from the vacuole, also PAO-insensitive. It is suggested that this involves the same aquaporin, either rendered directly ion-permeable, or signalling to activate an associated ion channel.

Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.

Preparation and Characterisation of Homogeneous Neurotoxin Type A from Clostridium botulinum

1. Large-scale production and purification of complexes between Clostridium botulinum neurotoxin and haemagglutinin have been achieved. 2. Haemagglutinin-free neurotoxic protein of the complexes was purified to high specific neurotoxicity by affinity chromatography, on p-aminophenyl beta-D-thiogalactopyranoside coupled to Sepharose 4B, followed by chromatography on DEAE-Sephacel. 3. The resultant neurotoxin was homogeneous on isoelectric focussing (pI = 6.3) and on dodecylsulphate/polyacrylamide gel electrophoresis under non-reducing conditions when its Mr was 1.4 X 10(5); after reduction two polypeptides (Mr = 9.9 and 5.5 X 10(4) were present. 4. On double-immunodiffusion gels, using antiserum against neurotoxin-haemagglutinin complex, the neurotoxin showed a single, sharp precipitin line that was immunologically distinct from a relatively non-toxic protein (Mr = 1.3 X 10(5), which co-purifies with the neurotoxin but is removed by the ion-exchange chromatography step. 5. Application of the neurotoxin to animals in vitro or in vivo produced near complete and irreversible blockade of neurotransmission. Botulinisation of rat leg muscles reduced spontaneous transmitter release; the amplitude of miniature end-plate potentials was altered from the normal 'bell-shaped" to a skewed distribution. 6. In normal muscle, a large transient increase in frequency of the miniatures was produced by beta-bungarotoxin. In contrast, with botulinised muscle the latter induced a much smaller increase in the absolute frequency; in addition, the mean amplitude was increased somewhat but the distribution remained skewed. The results show botulinisation of muscle modifies the action of beta-bungarotoxin.