Functional genomics and proteomics: application in neurosciences.

The sequencing of the complete genome for many organisms, including man, has opened the door to the systematic understanding of how complex structures such as the brain integrate and function, not only in health but also in disease. This blueprint, however, means that the piecemeal analysis regimes of the past are being rapidly superseded by new methods that analyse not just tens of genes or proteins at any one time, but thousands, if not the entire repertoire of a cell population or tissue under investigation. Using the most appropriate method of analysis to maximise the available data therefore becomes vital if a complete picture is to be obtained of how a system or individual cell is affected by a treatment or disease. This review examines what methods are currently available for the large scale analysis of gene and protein expression, and what are their limitations.

Basic properties of the sound-evoked post-auricular muscle response (PAMR).

One objective electrophysiological test for deafness involves presenting a brief acoustic stimulus to a subject and measuring the electrical activity evoked in the muscle located just behind the ear (the post-auricular muscle or PAM). Although this electrical response has been known for many years, it has been ignored by most clinicians and frequently misreported in the literature. This paper presents the fundamental properties of the PAM electrical response (the PAMR) and examines ways in which its measurement can be improved by altering the standard electrode position and filtering. The response consists of a simple bipolar compound action potential with a first peak latency of between 12.5 and 15 ms, depending on the stimulus intensity and PAM muscle tone. The largest recordings can be made with an active electrode over the PAM and with the reference electrode on the dorsal surface of the pinna. It can be obtained with click and tone-burst stimuli within 20 dB of the subjective detection threshold, can be evoked with tone-bursts between 500 Hz and 16 kHz and grows either linearly with the click level or approximately exponentially with the tone-burst level, reaching a maximum of as large as 250 microV pp in some subjects. It has a frequency spectrum mostly between 25 and 200 Hz. The response is often visible in raw recordings, with as few as 20 averages required for obtaining a stable waveform. There is very little amplitude and latency difference in stimulating the ear on the same side or opposite side to the recording electrodes and the binaurally evoked response is similar to the simple arithmetic sum of the waveforms obtained with monaural stimulation. The response latency and duration are longer in very young infants, but reach adult values by 12 months of age. In a companion paper, we describe a method of enhancing the PAMR using lateral eye movement (Patuzzi and O'Beirne, 1999a).

Effects of eye rotation on the sound-evoked post-auricular muscle response (PAMR).

One objective electrophysiological test for deafness involves presenting a brief acoustic stimulus to a subject and measuring the electrical activity evoked in the muscle located just behind the ear (the post-auricular muscle or PAM). We describe a method for enhancing this post-auricular muscle response (PAMR) using lateral eye movement, which increases both the tonic EMG activity in the PAM and the magnitude of the PAMR, and decreases response latency. EMG activity in most subjects tested (more than 30) increased almost instantly on rotation of the eyes, and thereafter grew more slowly with maintained lateral gaze, with the largest increase occurring with eye rotation towards rather than away from the measurement electrodes over the PAM. The EMG activity returned rapidly to near pre-rotation levels when the eyes were returned to the forwards position, with full recovery taking some minutes. While there was a similar increase and return of the PAMR amplitude with eye rotation, the time-course of these changes was somewhat different, largely because the EMG activity and the PAMR amplitude were not proportional. Rather the PAMR amplitude was a saturating function of EMG level, so that the PAMR response did not fall as markedly as the EMG when the eyes were returned to a forwards gaze, and the recovery of the PAMR amplitude to pre-rotation levels appeared to take longer. We discuss the neural mechanisms that may be responsible for this PAMR potentiation with eye movement and discuss its probable role in increasing variability in early studies which did not control for eye movement. We also discuss the utility of eye rotation in potentiating and stabilising the PAMR to allow its use in screening for deafness.

A correlation method for detecting the sound-evoked post-auricular muscle response (PAMR).

We have made detailed measurements of the sound-evoked post-auricular muscle response (PAMR) in four adults and two infants, in an attempt to understand the inter-relationships between sound level, potentiation of the PAMR with voluntary PAM contraction or eye rotation, electromyographic (EMG) noise, amplitude of the PAMR, and a correlation measure of the presence of the PAMR. We have found that the amplitude of the PAMR is a simple linear function of the decibel level of a monophasic click (0.1 ms duration), and that the PAMR amplitude is also a saturating power function of the level of tonic EMG. As a result, PAMR=PAM(o).SL. (EMG-EMG(noise))(2)/[(EMG-EMG(noise))(2)+beta(2)], where SL is the decibel level of a click above subjective threshold, PAM(o) is a parameter accounting for the differing PAMR amplitude across individuals or with altered electrode placement, EMG(noise) is the component of EMG not associated with PAMR potentiation, and beta determines the initial rate of growth of PAMR at low levels of PAM activation. We have also found that the correlation measure (C) of the PAMR follows a saturating power function of the signal-to-noise ratio (SNR=PAMR/EMG), with C=SNR(2)/(SNR(2)+delta(2)), where delta determines the onset of saturation in the correlation as a function of SNR. The combination of these two relationships means that correlation is a non-monotonic function of the EMG (PAM activation): it can be large for moderate levels of EMG, but small for high levels of EMG, because the PAMR amplitude saturates but the EMG does not. The correlation is a fast, convenient means of detecting the PAMR, whether using clicks or tone-bursts, and can be used effectively in adults or infants, as long as the reflex is moderately activated. This moderate activation is most effectively produced by eye rotation towards the recording electrodes.

Boltzmann analysis of CM waveforms using virtual instrument software.

We describe a modification to our technique for the rapid analysis of low-frequency cochlear microphonic (CM) waveforms in the basal turn of the guinea pig cochlea (Patuzzi and Moleirinho, 1998). The transfer curve relating instantaneous sound pressure in the ear canal to instantaneous receptor current through the outer hair cells (OHCs) is determined from the distorted microphonic waveform generated in the extracellular fluid near the hair cells, assuming a first-order Boltzmann activation curve. Previously, the analysis was done in real time using custom-built electronic circuitry. Here, the same task is performed numerically using virtual instrument software (National Instruments LabVIEW 4.1) running on a personal computer. The assumed theoretical function describing the CM waveform is Vcm = Voff + Vsat/[1 + exp[(Eo+Z.Po.sin(2pi f + phi(tot)))/kT]], where the six parameters are (i) a DC offset voltage (Voff); (ii) the frequency of the sinusoidal stimulus (f); (iii) the phase of the sinusoidal stimulus (phi(tot)); (iv) the maximal amplitude of the distorted microphonic signal (Vsat); (v) the sensitivity of the transduction process (Z); and (vi) the operating point on the sigmoidal transfer curve (Eo). The software obtains the least-squares fit to the CM waveforms by continuously deriving the six parameters at a speed of about one determination per second. The independent fitting of the frequency and phase allows the data to be analysed off-line from data previously recorded to tape (i.e. the frequency and phase of the microphonic response need not be known accurately beforehand). We present here an outline of the software we have used, and give an example of the changes which can be monitored using the technique (transient asphyxia). The method's advantages and limitations have been discussed in our previous paper. The virtual instrument described here is available from the authors on request.

Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression.

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.

Interactions between inositol trisphosphate receptors and fluorescent Ca2+ indicators.

Fura-2 and BAPTA were previously shown to be competitive antagonists of inositol trisphosphate (InsP3) receptors, but for practical reasons the analyses were performed at pH 8.3. We recently developed a scintillation proximity assay (SPA) for pure cerebellar InsP3 receptors which allows low affinity interactions to be characterized and is readily applicable to scarce or expensive ligands. In the present study, we use SPA to demonstrate that at pH 7.2, many of the commonly used fluorescent Ca2+ indicators reversibly displace 3H-InsP3 from its receptor and that they differ substantially in their affinities for the InsP3 receptor (IC50 = 6.5-137 microM). Recombinant type 1 InsP3 receptors expressed in Sf9 cells were used to examine 3H-InsP3 binding in cytosol-like medium: both fura-2 (IC50 = 796 +/- 86 microM) and Ca Green-5N (IC50 = 62 +/- 7 microM) completely inhibited the binding, but only in their Ca(2+)-free forms. Similar results were obtained with type 3 InsP3 receptors. We conclude that many Ca2+ indicators in their Ca(2+)-free forms compete with InsP3 for binding to its receptor, and that for Ca Green-5N the interaction occurs with sufficient affinity to significantly perturb physiological responses.

Scintillation proximity assay for E-, P-, and L-selectin utilizing polyacrylamide-based neoglycoconjugates as ligands.

In this study, a novel scintillation proximity assay (SPA) that uses radiolabeled soluble neoglycoconjugates as synthetic alternatives to the natural E-, P-, and L-selectin counterligands was developed. The neoglycoconjugates contained sialyl LewisX or sialyl LewisA attached via a three-carbon spacer to a poly[N-(hydroxyethyl)acrylamide] backbone, thus presenting the carbohydrates in a multivalent form. Selectin-ZZ fusion proteins were immobilized on anti-rabbit IgG-coated SPA beads via a rabbit IgG bridge. The neoglycoconjugate ligands bound to all three bead-immobilized selectins, with the highest binding levels apparent with E-selectin. Saturation binding studies with E-selectin revealed a complex interaction indicative of two or more binding affinities. The response to carbohydrate inhibitors was comparable in E-selectin assays that used either the neoglycoconjugates or the tritium-labeled HL60 cells as selectin counterligands. The incorporation of tyrosine sulfate groups into the backbone of the neoglycoconjugate resulted in enhanced binding avidity to both P- and L-selectin, indicating that the sulfate-containing neoglycoconjugates are viable synthetic mimics of the natural P- and L-selectin counterligands. The use of these radiolabeled neoglycoconjugates in conjunction with SPA results in a format ideally suited for the high-throughput screening for selectin antagonists. Furthermore, this approach can potentially be used to measure other low-avidity lectin-carbohydrate interactions.

Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2+ mobilization.

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly bound to multiple sites on InsP3 receptors and Ca2+ increased the binding by 190% +/- 10%; the half-maximal effect occurred when the Ca2+ concentration was 184 +/- 14 nM. In the absence of Ca2+, calmodulin caused a reversible, concentration-dependent (IC50 = 3.1 +/- 0.2 microM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect was similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 microM) inhibited the Ca2+ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways.

Noninvasive, real-time method for the examination of thymidine uptake events--application of the method to V-79 cell synchrony studies.

[14C]Thymidine uptake into V-79 hamster lung fibroblasts has been successfully demonstrated using a noninvasive, real-time method utilizing Cytostar-T scintillating microplates. These plates are standard format, tissue culture-treated, 96-well microplates with an integral scintillating base. The microplates permit the culture and observation of adherent cell monolayers. Biological activities of the cells can be studied by the provision of specific radiolabeled compounds. The biological activities of the adherent monolayer bring the specific radiolabel into proximity with the scintillating base and a scintillation signal is thereby generated. [14C]Thymidine incorporation on the microplates can be used to examine cell proliferation and cell cycle events. Using a combined mitotic shake-off/aphidicolin treatment to achieve synchronization, the thymidine incorporation activities of V-79 cells have been examined on Cytostar-T plates and correlated to traditional methods of determining incorporation. The method was further used to examine the effects of colcemid and olomoucine, both chemical inhibitors of cell proliferation, on synchronous populations of cells. The homogeneous detection format and the microplate nature of the method suggest a role for scintillating microplates in cell biology research and drug discovery.

An homogeneous assay for measuring the uptake and efflux of radiolabelled drugs in adherent cells.

We have developed an homogeneous assay to measure the uptake and efflux of [14C]adriamycin (doxorubicin hydrochloride) in human squamous lung carcinoma cells (SKMES-1), using 96 well scintillating microplates. The assay was also used to examine the effect of inhibitors of multidrug resistance in adriamycin resistant cells (SKMES-1/ADR). The effect of adriamycin on cell growth and viability was examined by continuous monitoring of the uptake of [14C]thymidine. The non-invasive nature of these assays, and the ease of use of the microplates, suggests a role in screens for, and characterisation of, novel chemotherapeutic or chemosensitizing agents.

Efficacy of a sonic toothbrush on inflammation and probing depth in adult periodontitis.

This single-blind, 8-week study compared the efficacy of a sonic toothbrush and a manual brush in 40 patients with adult periodontitis. Qualitative clinical indices and quantitative laboratory methods were used to monitor the periodontal status of 3 pockets 5 to 7 mm deep in each subject. Patients were randomly assigned either a sonic or manual toothbrush. The two groups were comparable with respect to age, gender, and anatomical location of the test sites. Data were collected from all sites at baseline and at 2, 4, and 8 weeks. Over the 8-week period, both groups showed significant improvements in the clinical indices used. Descriptive statistics indicated the sonic brush group had greater improvement than the manual group in the clinical parameters (gingival index, bleeding index, probing depth, and clinical attachment level). Gingival crevicular fluid (GCF) flow was significantly lower in the sonic brush group (P = 0.018). Considerable variation was present in the levels detected for both inflammatory cytokines tested, however, concentration of interleukin-1 beta was significantly lower in the GCF of sonic group patients (P = 0.05), while concentration of interleukin-6 was significantly reduced in both groups (P < or = 0.05) (t tests). Under these conditions, there is some evidence to suggest that the sonic toothbrush is more beneficial in resolving inflammation in patients with moderate periodontal disease.

Kinetic analysis of inositol trisphosphate binding to pure inositol trisphosphate receptors using scintillation proximity assay.

Inositol 1,4,5-triphosphate (InsP3) receptors are regulated by many intracellular signals including proteins and small messengers. By linking purified cerebellar InsP3 receptors to scintillation proximity assay beads, binding of radioligands can be measured without separation of bound from free ligand. InsP3 receptors assayed by scintillation proximity assay bound heparin with high affinity and stereoselectively bound InsP3 with similar affinity to cerebellar membranes. By rapidly freezing scintillation proximity assay reactions and then counting the frozen samples, both fast and slow components of [3H] InsP3 association and dissociation were identified. Our novel freeze-quench method in combination with conventional stopped-quench equipment and scintillation proximity assay allows the rapid kinetics of the interactions of pure receptors with their ligands to be resolved.

Xenovulene A, a novel GABA-benzodiazepine receptor binding compound produced by Acremonium strictum.

Xenovulene A, a novel inhibitor of benzodiazepine binding to the GABA-benzodiazepine receptor is produced by submerged fermentation of Acremonium strictum. It was isolated from the mycelium by solvent extraction and purified by chromatography on Sephadex LH-20 and octadecyl silica. The structure of xenovulene A was determined to be a novel oxygenated sesquiterpene containing a humulene moiety by interpretation of various spectroscopic data, especially from 2D NMR experiments. Xenovulene A inhibited binding of the benzodiazepine, flunitrazepam, with an IC50 of 40 nM in an in vitro assay using bovine synaptosome membrane preparations.

Shade selection for porcelain restorations.

This short paper describes the fundamental qualities of colour, applies these to the difficulties of choosing the correct shades for porcelain restorations and suggests a practical procedure for achieving a satisfactory shade match.

Two subcellular locations for peripheral-type benzodiazepine acceptors in rat liver.

Subcellular fractionation of rat liver by differential centrifugation showed the mitochondrial fractions to have the greatest enrichment of 'peripheral-type' benzodiazepine acceptor. Two peaks of acceptor sites were found on isopycnic density-gradient centrifugation, one peak (rho = 1.19 g/ml) corresponding to the peak of mitochondria as judged by marker enzyme distribution and by transmission electron microscopy, and the other peak (rho = 1.17 g/ml) which is not mitochondrial as judged by the lack of mitochondrial enzyme markers. Whereas the density of the mitochondrial acceptor was sensitive to sonication and was shown to have an outer-membrane location, the density of the non-mitochondrial acceptor was insensitive to sonication. The non-mitochondrial acceptor was shown not to be associated with Golgi, lysosomes, rough endoplasmic reticular microsomes, peroxisomes, or some types of plasma membranes, as judged by differences in the distribution of marker activities. No enrichment of benzodiazepine acceptor was found in the purified nuclear fraction. Both acceptors were shown to be peripheral-type high-affinity acceptors as judged by ligand specificities and by photoaffinity labelling.

Antiproliferative action of benzodiazepines in cultured brain cells is not mediated through the peripheral-type benzodiazepine acceptor.

The benzodiazepines, Ro 5-4864, diazepam, clonazepam, and also PK-11195, inhibited, at micromolar concentrations, the proliferation of rat C6 glioma and mouse neuro-2A neuroblastoma cells in culture. The cells possessed high levels of "peripheral-type" high-affinity benzodiazepine binding sites as judged by binding assays and displacement potencies. However, the different potencies and specificities of compounds for the antiproliferative actions and binding affinities for the binding site suggest that the antiproliferative actions were not mediated through the peripheral-type binding site. In support of this, these compounds have also been shown to inhibit proliferation of some nonneuronal cultured cell lines, e.g., mouse SP2/O-Ag 14 hybridoma and rat NCTC epithelial cells, which have no detectable high-affinity peripheral-type benzodiazepine binding sites.

The subcellular location in rat kidney of the peripheral benzodiazepine acceptor.

In rat kidney high-affinity binding sites for [3H]Ro-5-4864 and [3H]PK-11195 with the properties of the peripheral-type acceptor were found enriched in mitochondrial (M) and light-mitochondrial-lysosomal (L) fractions on differential centrifugation. When the combined M and L fractions were subjected to sucrose density gradient centrifugation, these binding sites were found enriched at a density of 1.155 g/ml coincident with a population of light mitochondria, whereas a population of heavier mitochondria (rho = 1.175 g/ml) had few or no binding sites. Transmission electron microscopy showed that whereas the heavier mitochondria appeared highly pure and intact, the lighter mitochondria appeared less intact and to be contaminated with vesicular structures. After fractionation of the light mitochondria and vesicles by centrifugation, both fractions showed the same ratio of [3H]Ro-4864 binding sites to monoamine oxidase activity consistent with the vesicles being of mitochondrial outer-membrane origin. Digitonin pre-treatment had no effect on the density of acceptor-rich fractions on sucrose density gradient centrifugation. However, pretreatment with succinate/iodophenylnitrophenylphenyltetrazolium (INT) perturbed equally the density of acceptor-rich fractions and mitochondrial marker enzymes. When mitochondrial fractions were subjected to sonication prior to density gradient centrifugation the binding sites were now found highly enriched in a much lighter fraction coincident with the monoamine oxidase activity and thus consistent with being outer-membrane vesicles. When a mitochondrial fraction was subjected to hypotonic treatment before assay no evidence for activation/unmasking of binding sites was found. The hypotonic treatment did not release any inhibitor of the binding sites. These results are consistent with the peripheral benzodiazepine acceptor having an outer-membrane location on a sub-population of rat kidney mitochondria. Those mitochondria showing high levels of the acceptor are either light mitochondria or appear more susceptible to osmotic damage than those mitochondria in which the acceptor is absent or at low levels.