Biomedical subjects

N F Sheppard

Publications and source records attributed to N F Sheppard.

6 recordsLinked to original sources

PubMed2001

Enzyme microgels in packed-bed bioreactors with downstream amperometric detection using microfabricated interdigitated microsensor electrode arrays.

In this article, we describe the use of pH- responsive hydrogels as matrices for the immobilization of two enzymes, glucose oxidase (GOx) and glutamate oxidase (GlutOx). Spherical hydrogel beads were prepared by inverse suspension polymerization and the enzymes were immobilized by either physical entrapment or covalent immobilization within or on the hydrogel surface. Packed-bed bioreactors were prepared containing the bioactive hydrogels and these incorporated into flow injection (FI) systems for the quantitation of glucose and monosodium glutamate (MSG) respectively. The FI amperometric detector comprised a microfabricated interdigitated array within a thin-layer flow cell. For the FI manifold incorporating immobilized GOx, glucose response curves were found to be linear over the concentration range 1.8-280 mg dL(-1) (0.1-15.5 mM) with a detection limit of 1.4 mg dL(-1) (0.08 mM). Up to 20 samples can be manually analyzed per hour, with the hydrogel-GOx bioreactor exhibiting good within-day (0.19%) precision. The optimized FI manifold for MSG quantitation yielded a linear response range of up to 135 mg dL(-1) (8 mM) with a detection limit of 3.38 mg dL(-1) (0.2 mM) and a throughput of 30 samples h(-1). Analysis of commercially produced soup samples gave a within-day precision of 3.6%. Bioreactors containing these two physically entrapped enzymes retained > 60% of their initial activities after a storage period of up to 1 year.

Aspergillus niger↗

PubMed1999

Mapping membrane potential transients in crayfish (Procambarus clarkii) optic lobe neuropils with voltage-sensitive dyes.

Voltage-sensitive dyes NK 2761 and RH 155 were employed (in conjunction with a 12 x 12 photodiode array) to study membrane potential transients in optic lobe neuropils in the eye stalk of the crayfish Procambarus clarkii. By this means we investigated a pathway linking deutocerebral projection neurons, via hemiellipsoid body local interneurons, to an unidentified target (most likely neurons processing visual information) in the medulla terminalis. Rapid (10- to 20-ms duration), transient changes in absorption with the characteristics of action potentials were recorded from the optic nerve and the region occupied by deutocerebral projection neurons after stimulation of the olfactory globular tract in the optic nerve and were blocked by 1 microM tetrodotoxin. Action potentials appeared to propagate to the glomerular layer of the hemiellipsoid body where synaptic responses were recorded from a restricted region of the hemiellipsoid body occupied by dendrites of hemiellipsoid body neurons. Action potentials were also recorded from processes of hemiellipsoid body neurons located in the medulla terminalis. Synaptic responses in the hemiellipsoid body and medulla terminalis were eliminated by addition to the saline of 500 microM Cd2+ or 20 mM Co2+, whereas the action potential attributed to branches of deutocerebral projection neurons in the hemiellipsoid body remained unaffected. Action potentials of hemiellipsoid body neurons in the medulla terminalis evoked postsynaptic potentials (50- to 200-ms duration) with an unidentified target in the medulla terminalis. Transient absorption signals were not detected in either the internal or external medulla nor were they recorded from other parts of the optic lobes in response to electrical stimulation of axons of the deutocerebral projection neurons. Functional maps of optical activity, together with electrophysiological and pharmacological findings, suggest that gamma-aminobutyric acid affects synaptic transmission in glomeruli of the hemiellipsoid body. Synapses of the olfactory pathway located in the medulla terminalis may act as a "filter," modifying visual information processing during olfactory stimulation.

Animals↗

PubMed1998

Functional organization of rat olfactory bulb glomeruli revealed by optical imaging.

The functional organization and synaptic physiology of olfactory bulb glomeruli were studied in rat in vitro slice preparations stained with the voltage-sensitive dye RH-155. Optical signals were recorded with a 100-element photodiode array at high temporal resolution. Pharmacological and ionic manipulations were used to investigate synaptic responses to stimulation of the olfactory nerve layer (ONL). ONL stimulation evoked a sodium-mediated compound action potential that propagated across the ONL and invaded individual glomeruli. This presynaptic volley evoked calcium-dependent synaptic responses the amplitudes of which were largest within the glomerular layer (GL); smaller amplitude responses were recorded in deeper layers of the olfactory bulb. Synaptic responses in the GL were attenuated by the non-NMDA ionotropic glutamate receptor antagonist CNQX; the residual component was suppressed by the NMDA glutamate receptor antagonist AP-5. The GABAA receptor antagonist bicuculline methiodide had little effect, whereas the GABAB receptor agonist baclofen dramatically attenuated ONL-evoked synaptic responses. The effects of baclofen were reversed by the GABAB receptor antagonist CGP35348. Paired-pulse depression of ONL-evoked synaptic responses in the GL was partially reversed by CGP35348. These findings suggest that olfactory nerve axons release glutamate to activate both NMDA and non-NMDA receptors on GL neurons, that GABAA receptor-mediated inhibition has little effect on these responses, and that GABAB receptor-mediated inhibition may act presynaptically on olfactory nerve axons to modulate their inputs to olfactory bulb neurons.

2-Amino-5-phosphonovalerate↗

PubMed1997

Evidence that calcium release-activated current mediates the biphasic electrical activity of mouse pancreatic beta-cells.

The electrical response of pancreatic beta-cells to step increases in glucose concentration is biphasic, consisting of a prolonged depolarization with action potentials (Phase 1) followed by membrane potential oscillations known as bursts. We have proposed that the Phase 1 response results from the combined depolarizing influences of potassium channel closure and an inward, nonselective cation current (ICRAN) that activates as intracellular calcium stores empty during exposure to basal glucose (Bertram et al., 1995). The stores refill during Phase 1, deactivating ICRAN and allowing steady-state bursting to commence. We support this hypothesis with additional simulations and experimental results indicating that Phase 1 duration is sensitive to the filling state of intracellular calcium stores. First, the duration of the Phase 1 transient increases with duration of prior exposure to basal (2.8 mM) glucose, reflecting the increased time required to fill calcium stores that have been emptying for longer periods. Second, Phase 1 duration is reduced when islets are exposed to elevated K+ to refill calcium stores in the presence of basal glucose. Third, when extracellular calcium is removed during the basal glucose exposure to reduce calcium influx into the stores, Phase 1 duration increases. Finally, no Phase 1 is observed following hyperpolarization of the beta-cell membrane with diazoxide in the continued presence of 11 mm glucose, a condition in which intracellular calcium stores remain full. Application of carbachol to empty calcium stores during basal glucose exposure did not increase Phase 1 duration as the model predicts. Despite this discrepancy, the good agreement between most of the experimental results and the model predictions provides evidence that a calcium release-activated current mediates the Phase 1 electrical response of the pancreatic beta-cell.

Animals↗

PubMed1997

Electrical conductivity of pH-responsive hydrogels.

The electrical conductivity of pH-responsive hydrogels based on cross-linked poly(2-hydroxyethyl methacrylate-co-dimethylaminoethyl methacrylate) copolymers has been studied as a function of pH over the range 5-10, for copolymers containing up to 20 mol% of the amine-containing comonomer. The conductivities of membranes equilibrated in buffered potassium chloride solutions were determined by measuring the electrical resistance of a membrane mounted between the chambers of a modified side-by-side diffusion cell. The conductivity, expressed as a fraction of the conductivity of the buffer in which the gels were equilibrated, ranged from 1% for the gels in the collapsed state to 70% for the most highly swollen gels. The observed results are qualitatively consistent with a proposed model in which the ion concentrations in the fluid phase of the gel are described by Donnan partitioning, and the ion mobilities by the free-volume theory of Yasuda. The results suggest that conductivity measurements may provide an alternative to diffusive transport studies for characterizing moderately swollen hydrogel membranes.

Cross-Linking Reagents↗

PubMed1995

Magnitude and modulation of pancreatic beta-cell gap junction electrical conductance in situ.

The parallel gap junction electrical conductance between a beta-cell and its nearest neighbors was measured by using an intracellular microelectrode to clamp the voltage of a beta-cell within a bursting islet of Langerhans. The holding current records consisted of bursts of inward current due to the synchronized oscillations in membrane potential of the surrounding cells. The membrane potential record of the impaled cell, obtained in current clamp mode, was used to estimate the behavior of the surrounding cells during voltage clamp, and the coupling conductance was calculated by dividing the magnitude of the current bursts by that of the voltage bursts. The histogram of coupling conductance magnitude from 26 cells was bimodal with peaks at 2.5 and 3.5 nS, indicating heterogeneity in extent of electrical communication within the islet of Langerhans. Gap junction conductance reversibly decreased when the temperature was lowered from 37 to 30 degrees C and when the extracellular calcium concentration was raised from 2.56 to 7.56 mM. The coupling conductance decreased slightly during the active phase of the burst. Activation of adenylate cyclase with forskolin (10 microM) resulted in an increase in cell-to-cell electrical coupling. We conclude that beta-cell gap junction conductance can be measured in situ under near physiological conditions. Furthermore, the magnitude and physiological regulation of beta-cell gap junction conductance suggest that intercellular electrical communication plays an important role in the function of the endocrine pancreas.

Animals↗