The neuron-level phenomena underlying cognition and consciousness: synaptic activity and the action potential.

An unusual property of the neuron is its capability for cell-to-cell communication via synapses, known to be the neuron-level "protophenomenon" underlying the brain-level "real phenomenon" of cognition. The temporal synchronization of such synaptic activity is the leading candidate for explaining "cognitive binding" and therefore the unity of mind. An equally-unusual property of the neuron is the action potential, the means by which the neuron sends a signal down the axon. Although infrequently noted by researchers in relation to consciousness, signal propagation within the neuron entails the momentary permeability of the neuronal membrane, allowing a massive influx of charged ions into the cellular interior. Such openness to the extracellular world is arguably the protophenomenon of neuronal "sentience," literally, feeling the charge-state of the electrochemical environment. Sensitivity to the external pH is a common feature of all living cells, but is greatly amplified during the neuron's action potential. Synchronization of the action potentials of the same neurons that are involved in cognitive binding is the likely mechanism by which the sentience of individual neurons is coordinated into the brain-level phenomenon of subjective awareness. I conclude that a proper understanding of the permeability of the neuronal membrane during the action potential is as important for consciousness studies as is a proper understanding of synaptic transmission for the explication of the cognition made possible by neurons.

Biologic mechanisms of 89SrCl2 incorporation into type I collagen during bone mineralization.

UNLABELLED: 89SrCl2 is currently used as a palliative treatment for painful osseous metastases associated with an osteoblastic reaction in bone. However, the underlying biologic mechanism by which 89SrCl2 accumulates at these lesions and mediates palliation remains unclear. The aim of this study was therefore to elucidate this mechanism. METHODS: An in vitro cell biologic model, incorporating the MC3T3-E1 murine osteoblast cell line, was established to replicate the process of collagen production and mineralization. Experiments were performed to investigate the cellular association of 89SrCl2 and 45CaCl2 with both MC3T3-E1 cells and the PC-3 human prostate adenocarcinoma cell line. RESULTS: No evidence of intracellular localization of 89SrCl2 or 45CaCl2 was found for either cell line. Localization of radiolabel was seen to be associated with MC3T3-E1 cells but only in cultures that had undergone both differentiation and mineralization. The association of 89SrCl2 was inhibited by the alkaline phosphatase inhibitor levamisole, and extracellular localization of 89SrCl2 was confirmed by microautoradiography. CONCLUSION: 89SrCl2 acts as a calcium mimic and, as such, becomes associated with the collagen matrix produced by the MC3T3-E1 cells during collagen mineralization.

Simulating consciousness in a bilateral neural network: "nuclear" and "fringe" awareness.

A technique for the bilateral activation of neural nets that leads to a functional asymmetry of two simulated "cerebral hemispheres" is described. The simulation is designed to perform object recognition, while exhibiting characteristics typical of human consciousness-specifically, the unitary nature of conscious attention, together with a dual awareness corresponding to the "nucleus" and "fringe" described by William James (1890). Sensory neural nets self-organize on the basis of five sensory features. The system is then taught arbitrary symbolic labels for a small number of similar stimuli. Finally, the trained network is exposed to nonverbal stimuli for object recognition, leading to Gaussian activation of the "sensory" maps-with a peak at the location most closely related to the features of the external stimulus. "Verbal" maps are activated most strongly at the labeled location that lies closest to the peak on homologous sensory maps. On the verbal maps activation is characterized by both excitatory and inhibitory Gaussians (a Mexican hat), the parameters of which are determined by the relative locations of the verbal labels. Mutual homotopic inhibition across the "corpus callosum" then produces functional cerebral asymmetries, i.e., complementary activation of homologous "association" and "frontal" maps within a common focus of attention-a nucleus in the left hemisphere and a fringe in the right hemisphere. An object is recognized as corresponding to a known label when the total activation of both hemispheres (nucleus plus fringe) is strongest for that label. The functional dualities of the cerebral hemispheres are discussed in light of the nucleus/fringe asymmetry.

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.

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.

Intermanual transfer of proximal and distal motor engrams in humans.

We studied intermanual motor transfer for right-to-left or left-to-right direction of transfer between either proximal or distal upper extremity muscle groups. The influence of previously acquired motor engrams (original learning, OL) on learning efficiency of the contralateral side (transfer learning, TL) was examined in 26 right-handed healthy subjects. The task consisted of the drawing of meaningless figures. During TL, OL figures had to be reproduced as vertical mirror reversals. Data revealed a benefit for right-to-left but not left-to-right direction of transfer for time to complete a figure as well as a left-to-right transfer benefit for spatial motor precision. Furthermore, a benefit for intermanual transfer of training between proximal but not distal muscle groups was found when movement time to complete a figure was evaluated. Of special interest was the observation of a disadvantage due to prior contralateral learning for performance at right distal effectors. The asymmetrical transfer benefits with respect to side are in line with previous findings and support the proficiency model and the cross-activation model. Results further showed that intermanual transfer of training might differ with respect to muscle group involvement and suggest that, although primarily facilitating, previous opposite hand training may lead to inhibitory influences on subsequent contralateral reproduction.

The cerebral hemispheres and neural network simulations: design considerations.

The conclusions concerning hemispheric specializations based on neural network simulations, which were previously reported by Kosslyn, Chabris, Marsolek, and Koenig (1992), are shown not to be valid. Differences in network performance on tasks said to be "categorical" and "coordinate spatial" in nature were due to imbalances in the input stimuli and cannot, in principle, be related to differences in performance on such tasks in human subjects. The use of truth tables and correlation coefficients in the design of neural networks is discussed.

Direct measurement of the binding of RAS to neurofibromin using a scintillation proximity assay.

Protein-protein interactions are of major importance in many cellular processes. When no enzymic activity is involved, assays for direct binding are required. One such example is the relatively weak interaction between oncogenic Ras and the GTPase-activating protein neurofibromin (NF1). The complex between the catalytic domain of NF1 and the GTP-form of oncogenic Ras protein dissociates rapidly; hence, equilibrium binding must be quantitated. Scintillation proximity assay (SPA) technology, a radioisotopic technique that requires no separation step, was used to characterize this interaction. Leu-61 Ras complexed with [3H]GTP was generated by nucleotide exchange in the presence of a GTP-regenerating system. A SPA signal was obtained when radiolabeled Ras was mixed with NF1 fused with glutathione S-transferase (GST), anti-GST, and protein A-coated SPA beads. This signal was abolished when any of the components were omitted and also by the addition of NaCl, which potently reduces the affinity of interaction between Ras and NF1. The neutralizing anti-Ras monoclonal antibody Y13-259 and the detergent n-dodecyl maltoside, a specific inhibitor of NF1 catalytic activity, both abolished the SPA signal from the NF1/Ras assay but neither affected a control SPA signal in which a [3H]GTP.GST-Ras fusion protein was bound to protein A-coated SPA beads. This technology could be readily extended to the measurement of other protein-protein interactions and could form the basis for high-throughput screens for the discovery of novel therapeutic agents.

Hemispheric cooperation in visuospatial rotations: evidence for a manipulation role for the left hemisphere and a reference role for the right hemisphere.

A survey of mental rotation strategies in 210 normal subjects showed a strong tendency for right-handers to prefer rotating an object on the right and vice versa for left-handers. The differential functioning of the cerebral hemispheres during mental rotation was then assessed in 42 subjects by means of tachistoscopic presentation of two geometrical figures separately to the left and right visual fields--one of which was gravitationally stable and the other unstable. Performance was better when the unstable object was presented to the right visual field and the stable object to the left. This finding is interpreted as indicating more efficient hemispheric cooperation when the active manipulation of a mental image is performed by the left hemisphere, while the reference role is carried out by the right hemisphere.

Interactions of substrates and inhibitors with a family of tethered HIV-1 and HIV-2 homo- and heterodimeric proteinases.

Genes were constructed to encode single-chain tethered human immunodeficiency virus HIV-1/HIV-1 and HIV-2/HIV-2 homodimeric proteinases and two HIV-1/HIV-2 heterodimers which differed in the nature of the interface strands. All four constructs under the control of a heat-inducible promoter were expressed in E. coli and the resultant proteinases were purified therefrom. Kinetic parameters (Km, kcat and kcat/Km) were derived for the interaction of the tethered homo and heterodimeric proteinases with two distinct substrates at a variety of pH values. All four enzymes were comparably active toward one substrate. With the second substrate at pH 4.7, the kcat/Km value was best for the HIV-1/1 tethered homodimer, 15-fold lower for the two heterodimeric proteinases, and was reduced by an additional 6-fold for the HIV-2/2 homodimer. From the Ki values determined for the interactions of the four tethered dimer proteinases with a systematic series of synthetic inhibitors, a parallel trend was observed. Whereas several inhibitors were equipotent against all four enzymes, two were discriminatory in that they inhibited strongly the HIV-1/1 homodimer and the two heterodimeric proteinases but had little effect on the HIV -2/2 tethered homodimer (or its untethered wild-type counterpart from HIV-2). The significance of these findings for active site interaction with HIV-proteinases is considered.

The dynamics of cerebral dominance during unilateral limbic seizures.

An apparent discrepancy exists between the remarkable language competence of the isolated right cerebral hemisphere in some split-brain patients and the lack of language competence in some aphasic patients in whom only the left cerebral hemisphere has been damaged. This has led to a revival of the idea that the dominant cerebral hemisphere inhibits the potential functioning of its partner. We tested this model of interhemispheric inhibition in four patients with unilateral limbic seizures (three left-sided, one right-sided). While seizure activity was monitored with bilaterally implanted stereotactic EEG depth electrodes or bilateral foramen ovale EEG electrodes, the patients were tested in lateralized hemisphere-specific tachistoscopic recognition experiments. In two patients performance was correlated with the electrical pattern during prolonged unilateral subclinical limbic status epilepticus, and in two patients performance and intracranial electrical activity were compared before, immediately after and during the recovery phase of left limbic complex partial seizures with postictal aphasia. Three main findings were obtained. (i) Focal unilateral limbic seizure activity in the depth interferes with cognitive functions, even when unnoticed clinically or undetectable on the surface EEG, provided that epileptiform EEG activity lasts for several seconds. (ii) This interference is specific to the cognitive functions associated with the 'dominant' processing of the discharging hemisphere. (iii) Seizure-induced impairment of the 'dominant' functions of the discharging hemisphere is associated with improvement of performance of the same function by the other hemisphere. Although each patient's clinical and experimental situation differed, the converging results support the hypothesis of hemispheric interaction based upon functional inhibition for verbal processing in patients with a left hemispheric focus. The reverse situation, namely facial processing in a single case with a right hemisphere focus, remains inconclusive.

Random-number generation and the menstrual cycle: preliminary evidence for a premenstrual alteration of frontal lobe functioning.

20 women (M age = 30.4 yr.) were given the "Mental Dice Task" (randomization of the numbers from 1 to 6) once during the preovulatory and once during the premenstrual phases of their menstrual cycles. In addition, for both test sessions a premenstrual-symptom score was assessed reflecting self-rated severity of cognitive, affective, and somatic complaints during the preovular and the premenstrual phases. In comparison to series of real dice throws, the Mental Dice sequences of all the subjects showed a relative lack of repetitions and an excess of counting at both times of testing. Counting bias was significantly enhanced in the sequences generated during the premenstrual testing and the size of this bias was positively correlated with the self-rated severity of premenstrual symptoms. Pronounced counting despite the instruction to randomize was interpreted as reflecting decreased ability to suppress task-irrelevant cues and indicating a relative impairment of frontal lobe functioning. Resistance of the Mental Dice Task towards expectation biases renders a sociopsychological interpretation of these results improbable. We conjecture that, like other cognitive processes, frontal lobe functions also fluctuate with hormonal changes.

Lateralized lexical decisions and the effects of hemifield masks: a study of interhemispheric inhibition and release.

A lateralized tachistoscope experiment using 40 normal, right-handed men tested the predictions of three different theories of interhemispheric interactions. Lexical decision for function words was measured for each visual field under several conditions of simultaneous stimulation in the contralateral hemifield. When a nonsense word was presented simultaneously to the contralateral hemifield, performance decreased, especially for words in the weaker field, an effect which is inconsistent with the predictions of the direct access model. When the contralateral nonsense word was immediately masked, there was a significant improvement in performance over the nonmasked condition. Analysis of the masking effect showed that the data are inconsistent with a callosal relay model, but are consistent with a model of transcallosal inhibition.

Hemispheric dissociation in judging semantic relations: complementarity for close and distant associates.

In two lateralized tachistoscopic experiments, we presented (i) pairs of nouns with close or distant semantic associations or (ii) pairs of nouns which were randomly matched and later rated by the subjects as to their semantic distance. In both experiments, words presented to the right visual field were more frequently judged as semantically close in meaning than words presented to the left visual field (LVF), whereas words presented to the LVF were more frequently judged as semantically distant. The results are discussed in relation to hemispheric language functions and current models of cerebral laterality.

Kinetic parameters for the generation of endothelins-1,-2 and -3 by human cathepsin E.

The specific conversion of human endothelin (ET) precursors big ET-1, big ET-2 and big ET-3 into their respective ET by cathepsin E was examined. Comparable pH optima were obtained for ET-1 and ET-2 generation, whereas effective conversion of big ET-3 into ET-3 necessitated a lower pH value. Determination of kinetic parameters (Km, kcat.) for all three conversions indicated that the precursors were efficiently bound by cathepsin E. The significance of the values obtained for the catalytic-centre activities and the effect of a specific inhibitor are discussed.

A simple method for measurement of phosphoramidon-sensitive endothelin converting enzyme activity.

We have developed a rapid and convenient assay for measurement of the action of endothelin (ET) converting enzyme (ECE) using the scintillation proximity assay (SPA) principle. On incubation of [125I]big ET-1 at 37 degrees C for 0.5-6 hr with an enzyme preparation, the reaction was terminated by the addition of an ET-1-specific antibody formulated in a buffer designed to shift the pH to alkaline. The antibody was allowed to come to equilibrium for 1 hr at room temperature and the amount of ET-1 produced, detected in a single step by the addition of protein A SPA beads. Using this assay, ECE activities of enzyme preparations obtained from porcine cultured endothelial cells and rat lung were clearly detected. These activities were inhibited by phosphoramidon in a concentration-dependent manner. The SPA based assay is homogeneous requiring no separation steps and takes a half day to complete. This method is therefore suitable for the high throughput screening of potential ECE inhibitors.

Generation of human endothelin by cathepsin E.

Highly-purified cathepsin D processed human big endothelin1-38 into endothelin-like fragments but did not appear to generate endothelin1-21 under the conditions employed. By contrast, human cathepsin E specifically cleaved human big endothelin into endothelin1-21 and the C-terminal fragment under identical conditions but did not degrade either product further.