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Biomedical subjects

R A Pearce

Publications and source records attributed to R A Pearce.

At least 19 recordsLinked to original sources
PubMed2006

An isoflurane- and alcohol-insensitive mutant GABA(A) receptor alpha(1) subunit with near-normal apparent affinity for GABA: characterization in heterologous systems and production of knockin mice.

Volatile anesthetics and alcohols enhance transmission mediated by gamma-aminobutyric acid type A receptors (GABA(A)Rs) in the central nervous system, an effect that may underlie some of the behavioral actions of these agents. Substituting a critical serine residue within the GABA(A)R alpha(1) subunit at position 270 with the larger residue histidine eliminated receptor modulation by isoflurane, but it also affected receptor gating (increased GABA sensitivity). To correct the shift in GABA sensitivity of this mutant, we mutated a second residue, leucine at position 277 to alanine. The double mutant alpha(1)(S270H,L277A)beta(2)gamma(2S) GABA(A)R was expressed in Xenopus laevis oocytes and human embryonic kidney (HEK)293 cells, and it had near-normal GABA sensitivity. However, rapid application of a brief GABA pulse to receptors expressed in HEK293 cells revealed that the deactivation was faster in double mutant than in wild-type receptors. In all heterologous systems, the enhancing effect of isoflurane and ethanol was greatly decreased in the double mutant receptor. Homozygous knockin mice harboring the double mutation were viable and presented no overt abnormality, except hyperactivity. This knockin mouse line should be useful in determining which behavioral actions of volatile anesthetics and ethanol are mediated by the GABA(A)Rs containing the alpha(1) subunit.

Animals↗
PubMed2006

Distribution of airborne microorganisms in commercial pork slaughter processes.

The objective of this research was to determine the prevalence and distribution of airborne bacterial contamination, with particular reference to Escherichia coli and Salmonella, at a number of stages in a pork slaughtering plant. Air samples (impaction and sedimentation) were recovered from seven locations before and during operations in a commercial pork processing plant. Aerobic mesophilic bacteria, E. coli counts and the incidence of Salmonella in the air were determined. Most sample locations which provided high impaction counts also provided high sedimentation counts. Before commencement of operations, there were no significant differences in aerobic mesophilic bacteria obtained from the sample locations. However, within 2 h of the commencement of operations, aerobic mesophilic bacteria in the wet room (3.14 log10 cfu/m3) were significantly higher (P < 0.05) than those in the clean room (2.66 log10 cfu/m3) and chiller (2.34 log10 cfu/m3). By the afternoon, similar aerobic mesophilic bacteria counts were recovered in the wet and clean rooms, although counts in both of these areas were significantly higher (P > 0.05) than in the chiller. In general there were no significant differences in E. coli counts between rooms (wet room, clean room and chiller) and these did not increase during the production day. Salmonella were detected at the locations of the dehairing and evisceration operations. Aerobic mesophilic bacteria in the air within the abattoir increased as production proceeded. In addition the air within the abattoir contained organisms such as Salmonella and E. coli. Positive correlations (P < 0.05-P < 0.001) between impaction and sedimentation samples were found suggesting that air may be an important source of carcass contamination.

Abattoirs↗
PubMed2005

Excision vs sponge swabbing - a comparison of methods for the microbiological sampling of beef, pork and lamb carcasses.

AIMS: The aim of this research was to compare excision sampling with polyurethane and cellulose acetate sponge swabbing for the recovery of total viable counts and Enterobacteriaceae on meat carcasses. METHODS AND RESULTS: Two sample types were used to compare the methods: (i) individual samples, taken from four sites on each carcass and (ii) composite samples, created by pooling the samples from four sites from an additional set of carcasses. When the polyurethane sponge and excision method were compared for individual sites, there were no significant differences in bacterial recovery on beef and pork carcasses and on two of four sites on lamb carcasses. However, when samples from each site were pooled, the excision method was more efficient than either swabbing method across the three animal species. CONCLUSIONS: Sampling using the polyurethane sponge represents an equivalent alternative method to excision for the bacteriological sampling of carcass surfaces which is nondestructive and less labour intensive. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the scientific basis for using sponge swabbing instead of excision in compliance with 2001/471/EC.

Animals↗
PubMed2004

Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro.

BACKGROUND: Propofol (2,6-diisopropylphenol) is a widely used general anaesthetic that modulates gamma-aminobutyric acid type A (GABA(A)) receptors, the major inhibitory neurotransmitter receptor in the brain. Previous studies have found that the concentration of propofol that is required to affect synaptic inhibition in brain slices is much higher than the free concentration that is achieved clinically and that modulates isolated receptors. We tested whether this is accounted for by slow equilibration in brain tissue, and determined the concentration that must be applied to achieve appropriate brain levels. METHODS: Rat brain slices 300-microm thick were placed in a solution of 100 microM propofol in artificial cerebrospinal fluid for times ranging from 7.5 to 480 min. Concentrations in these slices were measured by HPLC to determine diffusion and partition coefficients. Electrophysiological measurements of the rate at which effects of 5 microM propofol developed were compared with the calculated rate of increase in tissue concentration. RESULTS: The diffusion coefficient was approximately 0.02x10(-6) cm2 s(-1), and the brain:artificial cerebrospinal fluid partition coefficient was 36. Diffusion times in brain slices agreed well with time course measurements of propofol-induced depression of synaptic responses, which continued to increase over 5 h. This depression was reversed by blocking GABA inhibition with picrotoxin (100 microM). CONCLUSIONS: Propofol does enhance inhibition in brain slices at a concentration of 0.63 microM in the superfusate, which produces brain concentrations corresponding with those achieved in vivo, but equilibration requires several hours. It is likely that slow diffusion to GABA receptors accounts for the high concentrations (>10 microM) that were needed to depress evoked responses in previous investigations.

Anesthetics, Intravenous↗
PubMed2004

Studies to determine the critical control points in pork slaughter hazard analysis and critical control point systems.

Aerobic mesophilic counts (AMC), coliform (CC) and coliform resuscitation counts (CRCs) were obtained by swabbing 50 cm(2) areas at three sites (ham, belly and neck) on pig carcasses, after each of seven stages of the slaughter/dressing process (bleeding, scalding, dehairing, singeing, polishing, evisceration and chilling). In most cases, there were no statistical differences (P>0.05) among the counts derived by these three methods. Reductions in counts at individual sites were observed after scalding (3.5 log(10) cfu cm(-2)), and singeing (2.5 log(10) cfu cm(-2)). Increases in counts at individual sites were observed after dehairing (2.0 log(10) cfu cm(-2)) and polishing (1.5 log(10) cfu cm(-2)). The incidence of Salmonella on pig carcasses was also obtained by swabbing the outside surfaces of 100 half carcasses. Information on the incidence of Salmonella in scald tank water (108 samples) was also investigated. Carcass swabs and scald tank water were examined for the presence of Salmonella using standard enrichment methods. Salmonella were detected on 31% of carcasses immediately after bleeding, 7% of carcasses immediately after dehairing and evisceration, and 1% of carcasses immediately after scalding. Serovars included Salmonella Typhimurium, Salmonella Hadar, Salmonella Infantis and Salmonella Derby. No Salmonella were recovered from samples of scald tank water. The impact of pig slaughter/dressing processes on carcass microbiology and their potential use as critical control points (CCPs) during pork production are discussed.

Abattoirs↗
PubMed2003

Effects of gamma2S subunit incorporation on GABAA receptor macroscopic kinetics.

GABA(A) receptors, the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are heteropentameric proteins. We are interested in understanding the contribution of the gamma subunit to the kinetic properties of GABA(A) receptors. Studies in Xenopus oocytes have suggested that co-expression of alpha1, beta2, and gamma 2S subunits results in the formation of both alpha beta and alpha betagamma receptors (Boileau et al. 2002a; Boileau et al., 1998). Here, we have used an excess of the gamma 2S subunit in transfections of HEK293 cells to bias expression toward alpha beta gamma-containing receptors. Using rapid application and whole cell patch clamp techniques, we found that incorporation of the gamma subunit eliminated the rapid phases of desensitization and accelerated deactivation, consistent with a proposed role of desensitization in slowing deactivation. In addition, alpha betagamma receptors had an increased GABA EC(50), reduced sensitivity to block by Zn(2+), and did not display outward rectification as compared to alpha beta receptors.

Animals↗
PubMed2003

Prevalence of Campylobacter within a swine slaughter and processing facility.

In this work, the occurrence of Campylobacter in a swine slaughter and processing facility was studied. Thirty composite carcass samples, representing 360 swine carcasses, were taken immediately after exsanguination, immediately after polishing, after the final wash, and after overnight chilling at 2 degrees C. Thirty matching composite rectal samples were also taken immediately after exsanguination, and 60 nonmatching individual colon samples were collected from the same lot of swine during evisceration. Also, 72 environmental samples were collected from equipment used in the slaughter operation (42 samples) and the processing operation (30 samples). Campylobacter was isolated by direct plating on Campy-Line agar (CLA) or Campy-Cefex agar (CCA), as well as by Bolton broth enrichment and subsequent inoculation onto CLA or CCA. For all four recovery methods combined, Campylobacter was detected on 33% (10 of 30) of the composite carcasses immediately after exsanguination, 0% (0 of 30) after polishing, 7% (2 of 30) immediately before chilling, and 0% (0 of 30) after overnight chilling. The pathogen was recovered from 100% (30 of 30) of the composite rectal samples and 80% (48 of 60) of the individual colon samples. Campylobacter was detected in 4.8% (2 of 42) and 3.3% (1 of 30) of the slaughter and processing equipment samples, respectively. The recovery rate achieved with direct plating on CLA was significantly higher (P < 0.05) than those achieved with the other three recovery methods. For the 202 isolates recovered from all of the various samples tested, Campylobacter coli was the predominant species (75%) and was followed by Campylobacter spp. (24%) and Campylobacter jejuni (1%). These results indicate that although Campylobacter is highly prevalent in the intestinal tracts of swine arriving at the slaughter facility, this microorganism does not progress through the slaughtering operation and is not detectable on carcasses after overnight chilling.

Abattoirs↗
PubMed2002

Washing and chilling as critical control points in pork slaughter hazard analysis and critical control point (HACCP) systems.

AIMS: The aim of this research was to examine the effects of preslaughter washing, pre-evisceration washing, final carcass washing and chilling on final carcass quality and to evaluate these operations as possible critical control points (CCPs) within a pork slaughter hazard analysis and critical control point (HACCP) system. METHODS AND RESULTS: This study estimated bacterial numbers (total viable counts) and the incidence of Salmonella at three surface locations (ham, belly and neck) on 60 animals/carcasses processed through a small commercial pork abattoir (80 pigs d(-1)). Significant reductions (P < 0.05) in bacterial numbers were noted at some stages of the slaughter/dressing process, i.e. the process of hair removal (scalding-dehairing and singeing) resulted in an approx. 4.5 log10 cfu cm(-2) decrease in bacterial numbers. A significant increase (P < 0.05) in bacterial numbers was observed after pre-evisceration washing. Final washing increased the bacterial counts to between 3.6 and 3.8 log10 cfu cm(-2) while chilling effected a small but statistically significant (P < 0.05) increase to between 4.5 and 4.7 log10 cfu cm(-2). The incidence of Salmonella on pigs at the farm was 27%, decreasing to 10% after preslaughter washing. However, stunning and bleeding effected a considerable increase in Salmonella contamination and the incidence after these operations was 50%, which was reduced to 0% during the scalding-dehairing process. CONCLUSIONS: Washing the live animals and subsequent carcasses with cold water is not an effective control measure but chilling may be used as a CCP. SIGNIFICANCE AND IMPACT OF THE STUDY: Recent changes in European Union legislation legally mandate HACCP in pork slaughter plants. This research will provide a sound scientific basis on which to develop and implement effective HACCP in pork abattoirs.

Abattoirs↗
PubMed2000

Networks of interneurons with fast and slow gamma-aminobutyric acid type A (GABAA) kinetics provide substrate for mixed gamma-theta rhythm.

During active exploration, hippocampal neurons exhibit nested rhythmic activity at theta ( approximately 8 Hz) and gamma ( approximately 40 Hz) frequencies. Gamma rhythms may be generated locally by interactions within a class of interneurons mediating fast GABA(A) (GABA(A,fast)) inhibitory postsynaptic currents (IPSCs), whereas theta rhythms traditionally are thought to be imposed extrinsically. However, the hippocampus contains slow biophysical mechanisms that may contribute to the theta rhythm, either as a resonance activated by extrinsic input or as a purely local phenomenon. For example, region CA1 of the hippocampus contains a slower class of GABA(A) (GABA(A,slow)) synapses, believed to be generated by a distinct group of interneurons. Recent evidence indicates that these GABA(A,slow) interneurons project to the GABA(A, fast) interneurons that contribute to hippocampal gamma rhythms. Here, we use biophysically based simulations to explore the possible ramifications of interneuronal circuits containing separate classes of GABA(A,fast) and GABA(A,slow) interneurons. Simulated interneuronal networks with fast and slow synaptic kinetics can generate mixed theta-gamma rhythmicity under restricted conditions, including strong connections among each population, weaker connections between the two populations, and homogeneity of cellular properties and drive. Under a broader range of conditions, including heterogeneity, the networks can amplify and resynchronize phasic responses to weak phase-dispersed external drive at theta frequencies to either GABA(A,slow) or GABA(A,fast) cells. GABA(A, slow) synapses are necessary for this process of amplification and resynchronization.

Algorithms↗
PubMed2000

Effects of halothane on GABA(A) receptor kinetics: evidence for slowed agonist unbinding.

Many anesthetics, including the volatile agent halothane, prolong the decay of GABA(A) receptor-mediated IPSCs at central synapses. This effect is thought to be a major factor in the production of anesthesia. A variety of different kinetic mechanisms have been proposed for several intravenous agents, but for volatile agents the kinetic mechanisms underlying this change remain unknown. To address this question, we used rapid solution exchange techniques to apply GABA to recombinant GABA(A) receptors (alpha(1)beta(2)gamma(2s)) expressed in HEK 293 cells, in the absence and presence of halothane. To differentiate between different microscopic kinetic steps that may be altered by the anesthetic, we studied a variety of measures, including peak concentration-response characteristics, macroscopic desensitization, recovery from desensitization, maximal current activation rates, and responses to the low-affinity agonist taurine. Experimentally observed alterations were compared with predictions based on a kinetic scheme that incorporated two agonist binding steps, and open and desensitized states. We found that, in addition to slowing deactivation after a brief pulse of GABA, halothane increased agonist sensitivity and slowed recovery from desensitization but did not alter macroscopic desensitization or maximal activation rate and only slightly slowed rapid deactivation after taurine application. This pattern of responses was found to be consistent with a reduction in the microscopic agonist unbinding rate (k(off)) but not with changes in channel gating steps, such as the channel opening rate (beta), closing rate (alpha), or microscopic desensitization. We conclude that halothane slows IPSC decay by slowing dissociation of agonist from the receptor.

Anesthetics, Inhalation↗
PubMed2000

Kinetic differences between synaptic and extrasynaptic GABA(A) receptors in CA1 pyramidal cells.

GABA(A)-mediated IPSCs typically decay more rapidly than receptors in excised patches in response to brief pulses of applied GABA. We have investigated the source of this discrepancy in CA1 pyramidal neurons. IPSCs in these cells decayed rapidly, with a weighted time constant tau(Decay) of approximately 18 msec (24 degrees C), whereas excised and nucleated patch responses to brief pulses of GABA (2 msec, 1 mM) decayed more than three times as slowly (tau(Decay), approximately 63 msec). This discrepancy was not caused by differences between synaptic and exogenous transmitter transients because (1) there was no dependence of tau(Decay) on pulse duration for pulses of 0.6-4 msec, (2) responses to GABA at concentrations as low as 10 microM were still slower to decay (tau(Decay), approximately 41 msec) than IPSCs, and (3) responses of excised patches to synaptically released GABA had decay times similar to brief pulse responses. These data indicate that the receptors mediating synaptic versus brief pulse responses have different intrinsic properties. However, synaptic receptors were not altered by the patch excision process, because fast, spontaneous IPSCs could still be recorded in nucleated patches. Elevated calcium selectively modulated patch responses to GABA pulses, with no effect on IPSCs recorded in nucleated patches, demonstrating the presence of two receptor populations that are differentially regulated by intracellular second messengers. We conclude that two receptor populations with distinct kinetics coexist in CA1 pyramidal cells: slow extrasynaptic receptors that dominate the responses of excised patches to exogenous GABA applications and fast synaptic receptors that generate rapid IPSCs.

Animals↗
PubMed2000

Interactions between distinct GABA(A) circuits in hippocampus.

Synchronous activity among synaptically connected interneurons is thought to organize temporal patterns such as gamma and theta rhythms in cortical circuits. Interactions between distinct interneuron circuits may underlie more complex patterns, such as nested rhythms. Here, we demonstrate such an interaction between two groups of CA1 interneurons, GABA(A,slow) and GABA(A,fast) cells, that may contribute to theta and gamma rhythms, respectively. Stratum lacunosum-moleculare (SL-M) stimuli that activate GABA(A,slow) inhibitory postsynaptic currents (IPSCs) in pyramidal cells simultaneously depress the rate and amplitude of spontaneous GABA(A,fast) IPSCs for several hundred milliseconds. This suppression has a similar pharmacological profile to GABA(A,slow) IPSCs, and SL-M stimuli elicit GABA(A,slow) IPSCs in interneurons. We conclude that GABA(A,slow) cells inhibit both pyramidal cells and GABA(A,fast) interneurons and postulate that this interaction contributes to nested theta/gamma rhythms in hippocampus.

Animals↗
PubMed2000

Rapid and direct modulation of GABAA receptors by halothane.

BACKGROUND: Hypotheses regarding the nature of channel modulation by volatile anesthetics have focused primarily on "membrane actions" of anesthetics and more recently on direct actions of volatile agents on receptor proteins themselves. With the recognition that many channels are subject to modulation by intracellular enzymes, such as protein kinases and phosphatases, and recent demonstrations that the activity of these modulators themselves may be altered by anesthetic agents, a third possibility has been suggested:-anesthetic actions on channels may be indirect, produced, for example, via direct effects on intracellular enzyme systems. METHODS: To determine the contribution of indirect versus direct modulation, the authors compared effects of the volatile anesthetic halothane on gamma-aminobutyric acid A receptors under two conditions: in the whole cell configuration with intact intracellular regulatory systems, and in the excised patch configuration, in which intracellular signaling systems have been disrupted. They also evaluated the effects of rapid application and withdrawal of anesthetic to determine the time course of onset and offset of the anesthetic actions on these channels. RESULTS: Characteristic changes in gamma-aminobutyric acid A receptor function occurred in excised patches as in whole cells, did not require alteration of receptor phosphorylation, and were rapid (onset and offset of anesthetic action occurred within milliseconds). CONCLUSIONS: These results are not consistent with indirect modulation but rather indicate that volatile agents modulate gamma-aminobutyric acid A receptors by direct action on the channel complex or surrounding lipid membrane.

Adenosine Triphosphate↗
PubMed2000

Is anesthesia caused by potentiation of synaptic or intrinsic inhibition? Recent insights into the mechanisms of volatile anesthetics.

Volatile anesthetics modulate synaptic (GABAA receptor-mediated) and intrinsic (K+ channel-controlled) neuronal inhibition. GABAA receptor activity is enhanced, leading to increased charge transfer and prolonged synaptic inhibition, and members of the two pore domain family of potassium channels are activated, leading to neuronal hyperpolarization and reduced excitability. These effects may underlie different components of the complex anesthetic state.

Anesthetics, Inhalation↗
PubMed1999

Dual actions of volatile anesthetics on GABA(A) IPSCs: dissociation of blocking and prolonging effects.

BACKGROUND: Volatile agents alter inhibitory postsynaptic currents (IPSCs) at clinically relevant concentrations, an action that is thought to make an important contribution to their behavioral effects. The authors investigated the mechanisms underlying these effects by evaluating the concentration dependence of modulation by enflurane, isoflurane, and halothane of IPSCs in rat hippocampal slices. METHODS: Action potential-independent gamma-aminobutyric acid(A) IPSCs (miniature IPSCs [mIPSCs]) were recorded from CA1 pyramidal neurons. The effects on mIPSC amplitude were used to distinguish between presynaptic (altered release) and postsynaptic (altered receptor response) actions of volatile agents. The concentration dependence of blocking and prolonging actions was compared among the volatile agents to determine whether a single modulatory process could account for both effects. RESULTS: The application of volatile anesthetics prolonged the decay and reduced the amplitude of mIPSCs in a dose-dependent manner. The effects on decay time for isoflurane and enflurane could not be distinguished. However, the blocking effect of enflurane was significantly greater than that of isoflurane at all concentrations. Despite the blocking effect, the net action of these agents was enhanced inhibition, because charge transfer was always significantly greater than control. Isoflurane, and to a lesser extent enflurane and halothane, caused a picrotoxin-sensitive increase in baseline noise. Moderate increases in mIPSC frequency were also observed for all agents. CONCLUSIONS: These results show that enflurane, isoflurane, and halothane reduce IPSC amplitude through a direct postsynaptic action. Furthermore, the concentration dependence of the actions of the agents reveals a dissociation between the effects on the amplitude and the time course of IPSCs, suggesting that distinct mechanisms underlie the two actions.

Algorithms↗
PubMed1998

Concentration measures of volatile anesthetics in the aqueous phase using calcium sensitive electrodes.

Volatile anesthetic concentrations have been difficult to measure, but are an important experimental parameter for in vitro studies of anesthetic actions. Calcium sensitive electrodes were investigated as a means of continuously monitoring anesthetic concentrations in artificial cerebrospinal fluids (ACSF). Anesthetic-induced Ca2+ electrode signals were compared at room (22 degrees C) and physiological (35 degrees C) temperatures. Electrophysiological measures of anesthetic effects on synaptic potentials provided a bioassay. Halothane and isoflurane produced negative changes in calcium electrode potentials which were linearly related to concentrations over a clinically useful range (0.5-1.5 MAC). Anesthetic-induced voltages persisted in nominally zero Ca2+ ACSF and even in deionized water. A good correlation (r>0.9) was found for calcium electrode measures of anesthetic concentration and synaptic response depression produced by halothane, at both 22 and 35 degrees C. These results support three conclusions: (1) calcium sensitive electrodes provide a useful measure of volatile anesthetic concentrations in aqueous solution. (2) Care must be taken when using these electrodes for Ca2+ concentration measurements, if a volatile anesthetic is also to be used, since the anesthetic could introduce an appreciable error (>50%). (3) A temperature change of 13 degrees C had surprisingly little effect on Ca2+ electrode responses or on synaptic depression produced by anesthetics.

Anesthetics, Inhalation↗
PubMed1998

The synaptic basis of GABAA,slow.

Although two kinetically distinct evoked GABAA responses (GABAA,fast and GABAA,slow) have been observed in CA1 pyramidal neurons, studies of spontaneous IPSCs (sIPSCs) in these neurons have reported only a single population of events that resemble GABAA,fast in their rise and decay kinetics. The absence of slow sIPSCs calls into question the synaptic basis of GABAA,slow. We present evidence here that both evoked responses are synaptic in origin, because two classes of minimally evoked, spontaneous and miniature IPSCs exist that correspond to GABAA,fast and GABAA,slow. Slow sIPSCs occur infrequently, suggesting that the cells underlying these events have a low spontaneous firing rate, unlike the cells giving rise to fast sIPSCs. Like evoked GABAA,fast and GABAA,slow, fast and slow sIPSCs are modulated differentially by furosemide, a subtype-specific GABAA antagonist. Furosemide blocks fast IPSCs by acting directly on the postsynaptic receptors, because it reduces the amplitude of both miniature IPSCs and the responses of excised patches to applied GABA. Thus, in the hippocampus, parallel inhibitory circuits are composed of separate populations of interneurons that contact anatomically segregated and pharmacologically distinct postsynaptic receptors.

Animals↗