Functional identification and monitoring of individual alpha and beta cells in cultured mouse islets of Langerhans.

The aim of the present study was to evaluate, by use of fluorescence microscopy and immunofluorescence stainings, the use of a fluorescent membrane potential sensitive probe as a means to identify and monitor changes in membrane potential of individual cell types in whole islets of Langerhans over time. Our work supports the use of the fluorescent probe bis-(1,3 dibutylbarbituric acid) trimethine oxonol (diBAC(4)(3)), in identification of single alpha and beta cells in the periphery of mouse pancreatic islets cultured on extracellular matrix. At a low extracellular glucose concentration (3 mM), heterogeneous staining of the islets was observed. Approximately 97% of the peripheral cells that stained brightly with diBAC(4)(3) were glucagon positive. Additional diBAC(4)(3) studies, demonstrated that an increase in glucose concentration from 3 to 10 mM is paralleled by repolarization of alpha cells and depolarization of beta cells. This suggests that reciprocity of glucagon and insulin release upon glucose stimulation is coupled to divergent changes in membrane potential of these cell types and supports the use of diBAC(4)(3) as a means to detect changes in secretion in both cell types.

Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy.

The cDNA for the green fluorescent protein (GFP) of Aequorea victoria has been expressed in transformed cells of Saccharomyces cerevisiae and the recombinant GFP isolated. Protonation and deprotonation of the cloned and purified GFP produced major effects on its spectral absorption characteristics with an increase in pH enhancing the fluorescence emission of the GFP more than twofold. Finally, molecular characterisation of GFP by fluorescence correlation microscopy in a minimal target volume of 1 fL yielded a translational diffusion coefficient (DT) of 8.7 x 10(-7) cm2.sec-1, equivalent to a Stokes radius of 2.82nm for a monodisperse globular protein of 27kDa.

Multiple conductances in the large K+ channel from Chara corallina shown by a transient analysis method.

The large conductance K+ channel in the tonoplast of Chara corallina has subconductance states (substates). We describe a method that detects substates by monitoring the time derivative of channel current. Substates near to the full conductance tend to have long durations and high probabilities, while those of smaller amplitude occur with less probability and short duration. The substate pattern is similar in cell-attached, inside-out and outside-out patches over a range of temperatures. The pattern changes at high Ca2+ concentration (10 mol m-3) on the cytoplasmic face of inside-out patches. One substate at approximately 50% of the full conductance is characterized by a high frequency of transitions from the full conductance level. This midstate conductance is not a constant proportion of the full conductance but changes as a function of membrane potential difference (p.d.) showing strong inward rectification. We suggest that the channel is a single pore that can change conformation and/or charge profile to give different conductances. The mean durations of the full conductance level and the midstate decrease as the membrane p.d. becomes more negative. Programs for analysis of channel kinetics based on an half-amplitude detection criterion are shown to be unsuitable for analysis of the K+ channel.

The k/na selectivity of a cation channel in the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive wheat species.

The characteristics of cation outward rectifier channels were studied in protoplasts from wheat root (Triticum aestivum L. and Triticum turgidum L.) cells using the patch clamp technique. The cation outward rectifier channels were voltage-dependent with a single channel conductance of 32 +/- 1 picosiemens in 100 millimolar KCl. Whole-cell currents were dominated by the activity of the cation outward rectifiers. The time- and voltage-dependence of these currents was accounted for by the summed behavior of individual channels recorded from outside-out detached patches. The K(+)/Na(+) permeability ratio of these channels was measured in a salt-sensitive and salt-tolerant genotype of wheat that differ in rates of Na(+) accumulation, using a voltage ramp protocol on protoplasts in the whole-cell configuration. Permeability ratios were calculated from shifts in reversal potentials following ion substitutions. There were no significant differences in the K(+)/Na(+) permeability ratios of these channels in root cells from either of the two genotypes tested. The permeability ratio for K(+)/Cl(-) was greater than 50:1. The K(+)/Na(+) permeability ratio averaged 30:1, which is two to four times more selective than the same type of channel in guard cells and suspension culture cells. Lowering the Ca(2+) concentration in the bath solution to 0.1 millimolar in the presence of 100 millimolar Na(+) had no significant effect on the K(+)/Na(+) permeability ratios of the channel. It seems unlikely that the mechanism of salt tolerance in wheat is based on differences in the K(+)/Na(+) selectivity of these channels.

Ion channels in the plasma membrane of Amaranthus protoplasts: one cation and one anion channel dominate the conductance.

This report details preliminary findings for ion channels in the plasma membrane of protoplasts derived from the cotyledons of Amaranthus seedlings. The conductance properties of the membrane can be described almost entirely by the behavior of two types of ion channel observed as single channels in attached and detached patches. The first is a cation-selective outward rectifier, and the second a multistate anion-selective channel which, under physiological conditions, acts as an inward rectifier. The cation channel has unit conductance of approx. 30 pS (symmetrical 100 K+) and relative permeability sequence K+ greater than Na+ much greater than Cl- (1:0.16:0.03): whole-cell currents activate in a time-dependent manner, and both activation and deactivation kinetics are voltage dependent. The anion channel opens for hyperpolarized membrane potentials, has a full-level conductance of approx. 200 pS and multiple subconductance states. The number of subconductances does not appear to be fixed. When activated the channel is open for long periods, though shuts if the membrane potential (Vm) is depolarized; at millimolar levels of [Ca2+]cyt this voltage dependency disappears. Inward current attributable to the anion channel is not observed in whole-cell recordings when MgATP (2 mM) is present in the intracellular solution. By contrast the channel is active in most detached patches, whether MgATP is present or not on the cytoplasmic face of the membrane. The anion channel has a significant permeability to cations, the sequence being NO3- greater than Cl- greater than K+ greater than Aspartate (2.04:1:0.18 to 0.09:0.04). The relative permeability for K+ decreased at progressively lower conductance states. In the absence of permeant anions this channel could be mistaken for a cation inward rectifier. The anion and cation channels could serve to clamp Vm at a preferred value in the face of events which would otherwise perturb Vm.

A diagnostic case involving an incisive canal cyst.

An incisive canal cyst was misdiagnosed as an endodontic lesion. The patient's chief complaint was pain on mastication with increasing intensity for the last 2 days. The single original periapical radiograph revealed a well-circumscribed radiolucency related to the apex of the maxillary right central incisor. The patient was referred with a request for endodontic therapy. A subsequent angled radiograph revealed an interroot location of the lesion, and pulp testing showed a normal response. The patient was then referred for surgical treatment. Following removal of the lesion, the histopathologic examination confirmed the diagnosis of an incisive canal cyst. One-year follow-up showed complete healing and maintained tooth vitality, and the patient was asymptomatic.

Hydrodynamic radius alone governs the mobility of molecules through plasmodesmata.

Various fluorescent molecular probes have been injected into the cytoplasm of nectary trichome cells of Abutilon striatum to ascertain the conductivity of the plasmodesmata. Most of the probes were based on fluorescein conjugated to a range of amino acids and peptides. The probes are not broken down by cytoplasmic enzymes during the period of observation. The results indicate that there are no specific effects of aromatic amino acids, either polar or hydrophobic types, on the conductivity of the Abutilon plasmodesmata, contrary to reports for other plants. The conductivity of the plasmodesmata in the trichomes is slightly greater than for any that have been studied in the tissues of other plants. It is proposed that in Abutilon the mobility of a probe is determined solely by the effective Stokes radius of the molecule, and that the radius of the molecule is governed by the molecular weight and, in particular, by the nature of the side groups in the peptide chain attached to the fluorochrome. Calculations are presented which indicate that channels between material in the plasmodesmatal annulus are the most likely route for the diffusion of the probes, and that the width of individual channels in the annulus is close to 3 nm.