Characterization of TASK-4, a novel member of the pH-sensitive, two-pore domain potassium channel family.

We report the primary sequence of TASK-4, a novel member of the acid-sensitive subfamily of tandem pore K(+) channels. TASK-4 transcripts are widely expressed in humans, with highest levels in liver, lung, pancreas, placenta, aorta and heart. In Xenopus oocytes TASK-4 generated K(+) currents displaying a marked outward rectification which was lost by elevation of extracellular K(+). TASK-4 currents were efficiently blocked by barium (83% inhibition at 2 mM), only weakly inhibited by 1 mM concentrations of quinine, bupivacaine and lidocaine, but not blocked by tetraethylammonium, 4-aminopyridine and Cs(+). TASK-4 was sensitive to extracellular pH, but in contrast to other TASK channels, pH sensitivity was shifted to more alkaline pH. Thus, TASK-4 in concert with other TASK channels might regulate cellular membrane potential over a wide range of extracellular pH.

Cytological effects of platelet-derived growth factor on mitochondrial ultrastructure in fibroblasts.

The goal of this study was to evaluate morphofunctional changes in mitochondrial ultrastructure after platelet-derived growth factor application in fibroblasts as an indicator of mitochondrial activation in processes like wound healing. NRK-49F fibroblasts were synchronized, incubated with PDGF (platelet-derived growth factor) and studied by electron microscopy. Volume density (Vv), numerical density (Nv) and surface density (Sv) were measured by stereological analysis. Application of PDGF on NRK-49F caused an increase in mitochondrial volume density by 57% and surface area of cristae per mitochondrion by 65%. The numerical density of the mitochondria was decreased in the PDGF-treated cells by 23%, but at the same time their mean volume was increased. Furthermore, the mitochondria had a complex and highly variable shape both in control and PDGF-treated cells, possibly indicating the existence of a mitochondrial reticulum. The results demonstrated that biochemically active membrane systems in fibroblast mitochondria are enlarged as a direct effect of small doses of platelet-derived growth factor and support the concept that this factor and related peptides serve as mitogens for connective tissue forming cells. Thus, in mitogenic processes like wound healing, the high energy demand of fibroblasts is provided by the increase of the inner surface of mitochondria.

The preparation and execution of saccadic eye and goal-directed hand movements in patients with Parkinson's disease.

The oculomotor and manual motor systems were studied in a two-segment movement task in a group of patients with Parkinson's disease and in age matched normal controls. In order to avoid reflexive motor movements the selection of the correct motor sequence was derived from the interpretation of symbolic (coloured) cues. The latencies and dynamics of eye and hand (pointing) movements performed during the first (fixed) movement segment were measured and the planning and execution processes were manipulated by varying the complexity of the second movement segment relative to the first (with regard to direction and amplitude). The results showed that the eye and hand movements made by patients with Parkinson's disease were not impaired in the initiation of the first movement segment. Interestingly, both Parkinson's patients and controls showed increased eye and hand reaction time latencies for the first movement when the second movement was in the direction opposite to the first. This indicates that the complexity of the second movement influences the execution of the first movement, and importantly that complexity affects motor initiation and execution processes in both normal subjects and in patients with Parkinson's disease. The execution of hand movements was found to be impaired in patients with Parkinson's disease as indicated by a reduced peak velocity of manual pointing responses when compared to age matched controls. By contrast, no differences were found in the dynamics of saccadic eye movements. This dissociation is consistent with the notion that the skeletomotor loop passes through the functionally corresponding portions of the basal ganglia independently of the oculomotor loop. Together, these results demonstrate that Parkinson's patients are able to generate multiple non-reflexive eye and hand movements and that the observed (manual) motor deficits are specific to the processes of motor execution.

Eye movements and spatial working memory in Parkinson's disease.

Mechanisms of spatial working memory and eye movement control were investigated in eight mild to moderate Parkinson's disease patients (PDs). Subjects were presented with a sequence of four targets which had to be memorized and then recalled by moving their eyes to fixate the four locations in the correct order. Two variations on this procedure were used in which either a different sequence of lights was presented on each trial, or an identical sequence of lights was repeated on each trial. In both conditions subjects made memory-guided eye movements in the dark, without any visual cues to eye movement accuracy or the locations of the previously illuminated lights. Analysis of the amplitude of the primary eye movement and final eye position for each step in the sequence showed that PDs made several discrete saccadic eye movements of reduced amplitude before reaching the final eye position (multi-stepping). When a novel target sequence had to be memorized on each trial, the final eye position reached by PDs for each location was also found to undershoot relative to controls. In contrast, when an identical sequence of targets was repeated on each trial, PDs' final eye position was found to be normal, although primary movement amplitudes were still reduced. PDs showed no multi-stepping and normal final eye position gain under conditions for which the target lights in the sequence were illuminated during movement execution. PDs also made an increased proportion of overt errors in target sequence recall. Parallel neuropsychological testing in PDs and controls revealed that error rates in the sequential memory-guided saccade task were significantly correlated with performance in a task thought to be sensitive to spatial working memory dysfunction. The findings suggest that short-term spatial memory representations are disrupted in the early stages of PD.

Conditioning pigeons to discriminate naturally lit insect specimens.

Pigeons (Columba livia) were trained on a visual discrimination task using a novel apparatus which enabled pinned specimens of insects, illuminated by natural daylight, to be presented under a pecking key transparent to ultraviolet light. Three birds showed evidence of learning to discriminate between sets of wasp and fly specimens. This response transferred to specimens of four hoverfly species, the strength of the response varying between the different hoverfly species. This conditioning technique offers a promising means of analysing mechanisms of visual processing in birds that are relevant to theories of the evolution of camouflage and mimicry.

Preparing to react in the absence of uncertainty: I. New perspectives on simple reaction time.

Almost from the inception of psychological enquiry into reaction time (RT), a broad distinction has been drawn between two types of process-serving performance in RT tasks, namely, the on-line processes initiated by the arrival of the imperative signal and preparatory processes, which precede the imperative signal and may contribute to performance efficiency. Restriction of attention to on-line processing fosters the conclusion that the processes serving simple RT are no more than a subset of those involved in choice reactions. If, however, latencies can be reduced by specific preparation then the certitudes of the simple reaction task may invest it with distinctive properties. The possibility of preparation being sensory as well as motor needs to be considered. Until recently, attempts to study preparedness have failed to make controlled comparisons across simple and choice RT. However, recent work has shown that simple reactions have distinctive characteristics that set them apart from minimal choice reactions. The optimization of simple RT seems to depend upon attention-demanding processes which are probably preparatory in nature. When attention has to be shared with a concurrent task, much of the normal advantage of the simple reaction is lost. Neuropsychological studies suggest that patients may be found with abnormally extended simple RT but spared choice reactions. In these patients, the simple reactions are prolonged relative to those of controls but less vulnerable to imposition of a secondary task. Evidence is beginning to accrue to the effect that some distinctive processing features of simple reactions may be mediated by activity in the frontal lobes.

An in vivo approach for the identification of acceptor sites for O-glycosyltransferases: motifs for the addition of O-GlcNAc in Dictyostelium discoideum.

To identify and analyze acceptor sequences for O-glycosylation, we have developed an in vivo system expressing short peptides as glutathione S-transferase fusion proteins in the eukaryotic host Dictyostelium discoideum. Using this approach, we show that a short peptide motif (PTVTPT), present in the D. discoideum cell-surface glycoprotein PsA, is sufficient as a signal for O-glycosylation, even when fused to a heterologous protein. Monosaccharide analysis and solid-phase protein sequencing showed that the modification is a single N-acetylglucosamine attached to threonine residues. This was further confirmed by electrospray-mass spectrometry. The O-linked glycosylation of both this peptide and authentic PsA presents the modB-dependent carbohydrate-specific epitope identified by the monoclonal antibody MUD50. Substitution of threonine by serine residues in this peptide also yields a glycosylated fusion protein which is modified with single N-acetylglucosamine residues, but not all of the serines are glycosylated.

DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum.

A protein accumulated in the cortical region of Dictyostelium discoideum cells proved to be a homologue of GTPase activating proteins that are responsible for the inactivation of ras in yeast and man. Elimination of this protein, DGAP1, by gene replacement resulted in an increased rate of growth of D. discoideum cells on bacterial lawns, and in the formation of aberrant, multi-tipped fruiting bodies. Overexpression of DGAP1 caused the cells to become multi-nucleated since chromosome segregation during mitosis was not reliably followed by cleavage of the cells. These results suggest that in D. discoideum, ras or a related small GTP-binding protein is involved in regulating growth based on the phagocytosis of bacteria, and in coupling activities of the cell cortex to the organization of spindle and asters in mitotic cells.

Perception of emotion from dynamic point-light displays represented in dance.

It is well known that biological motion, as produced by point-light displays on a human body, gives a good representation of the represented body-eg its gender and the nature of the task which it is engaged in. The question is whether it is possible to judge the emotional state of a human body from motion information alone. An ability to make this kind of judgment may imply that people are able to perceive emotion from patterns of movement without having to compute the detailed shape first. Subjects were shown brief video clips of two trained dancers (one male, one female). The dancers were aiming to convey the following emotions: fear, anger, grief, joy, surprise, and disgust. The video clips portrayed fully lit scenes and point-light scenes, with thirteen small points of light attached to the body of each dancer. Half the stimuli were presented the right way up, while half were inverted. The subjects' task was to judge which emotion was being portrayed. Full-body clips gave good recognition of emotionality (88% correct), but the results for upright biological-motion displays were also significantly above chance (63% correct). Inversion of the display reduced biological-motion (but not full-body) performance to close to chance but still significantly above chance. A space-time analysis of the motion of the points of light was carried out, and was related to the discriminability of the different emotions. Biological-motion displays, which convey no information while static, are able to give a rich description of the subject matter, including the ability to judge emotional state. This ability is disrupted when the image is inverted.

pDcsA vectors for strictly regulated protein synthesis during early development of Dictyostelium discoideum.

Two expression vectors have been constructed to express proteins exclusively in developing cells of Dictyostellium discoideum. In these Escherichia coli/D. discoideum shuttle vectors, proteins are synthesized under control of the promoter of the contact site A (csA) gene, which is efficiently suppressed during growth and becomes strongly activated during early development of D. discoideum. The pDcsA vectors appear to be valuable tools for the production of proteins that are not compatible with growth of D. discoideum cells.

Production and secretion of recombinant proteins in Dictyostelium discoideum.

We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system. The cellular slime mold Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface associated D. discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme beta-glucuronidase (GUS) from Escherichia coli was cell associated. Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium. The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection.

Visual perception of intentional motion.

A series of experiments were performed to investigate how motion sequences provide information about the intentional structure of moving figures or actors. Observers had to detect simulations of biologically meaningful motion within a set of moving letters. In the first two experiments a factorial design was used, with type of instruction as a between-subject factor and six movement parameters (number of items, speed and directness of target and distractors, and 'relentlessness' of target movement) as within-subject factor; in the final two experiments, the visibility of the goal towards which the target moved and the use of a tracking movement to distinguish the target were varied. In such displays search time increases with increasing number of stimuli. It was found that (a) the more direct the motion, the more likely it was to be interpreted as intentional; (b) intentional motion was much easier to detect when the target moved faster than the distractors than when it moved more slowly; (c) recognition of intentionality was impaired but not abolished if the goal towards which the target was moving was invisible; and (d) participants did not report intentional movement when the target was distinguished by brightness rather than the manner in which it moved. We argue that the perception of intentionality is strongly related to observers' use of conceptual knowledge, which in turn is activated by particular combinations of features. This supports a process model, in which intentionality is seen as the result of a conceptual integration of objective visual features.

How monkeys see others: Discrimination and recognition of monkeys' shape.

The two experiments described in this study address the question of the perceptual basis of species discrimination and body recognition in monkeys. Longtailed macaques (Macaca fascicularis) were trained to discriminate line drawings of different monkey bodies. The procedure consisted of a simultaneous discrimination between four images under continuous reinforcement. Social communication between the test animal and other group members during test sessions was almost unrestricted. In the first experiment all monkeys learned, within at least 7 sessions, to discriminate one monkey from other monkeys. Discrimination was invariant against transformations of size and rotation of the stimuli. A preference test for particular features resulted in a graded estimation of particular body features. Generalisation to different views of facial stimuli was demonstrated. In the second experiment the monkeys had to relearn a new association which involved a differentiation of the previously shown stimuli. After reaching the learning criterion it was shown that the same features as in the previous experiment were evaluated differently. The experiments generally support the view that perceptual mechanisms of the signal receiver are crucial for individual recognition. Results are discussed in contrast to a 'theory of mind' approach in primate cognition.

Pretranslational regulation of type I 5'-deiodinase by thyroid hormones and in fasted and diabetic rats.

In both humans and experimental animals, nutritional deprivation and systemic illness are associated with decreases in circulating T3 levels, T3 production, and type I 5'-deiodinase (5'DI) activity. In order to assess the regulation of 5'DI messenger RNA (mRNA) in these conditions, a solution hybridization assay was developed which utilized a complementary (cDNA) encompassing 92% of the 5'DI coding region. The administration of T3 to hypothyroid rats induced a 50-fold increase in 5'DI mRNA levels that preceded by 12 h a similar increase in 5'DI activity. Fasting for 48-72 h, but not 24 h, was associated with an approximate 50% decrease in 5'DI mRNA levels in liver, which preceded a decrease of similar magnitude in 5'DI activity. Fasting in hyperthyroid rats did not lower hepatic 5'DI mRNA levels or activity, nor did fasting attenuate the stimulatory effects of T3 administration on these parameters. In diabetic animals 5'DI mRNA levels and activity were markedly reduced. These results demonstrate that regulation of 5'DI by thyroid hormones and in fasting and diabetes mellitus is exerted principally at the pretranslational level. The relatively late occurrence of decreases in 5'DI mRNA levels and activity observed in nutritional deprivation suggests that these changes are secondary to, rather than causative of, the decreased serum and tissue T3 levels noted in these conditions.

Motion as a natural category for pigeons: Generalization and a feature-positive effect.

Three groups of pigeons were trained with a modified discriminative autoshaping procedure to discriminate video images of other pigeons on the basis of movement. Birds of all groups were shown the same video images of other pigeons, which were either moving or still. The group to whom food was presented only after moving images learned the discrimination very quickly. A second group, to whom food was given only after still images, and a pseudocategory group, to whom food was presented after arbitrarily chosen stimuli, showed no evidence of discrimination during acquisition training. Extinction conditions led to clear differences in peck rates to moving and still images in the second group but not in the pseudocategory group. The result is related to the feature-positive effect. Generalization tests showed that the discrimination performance was based on visual features of the stimuli but was invariant against changes of size, perspective, brightness, and color. Furthermore, discrimination was maintained when novel images of pigeons under different viewing angles and seven other types of motion categories were presented. It is argued that the discrimination is based not on a common motion feature but on motion concepts or high-order generalization across motion categories.

Action categories and the perception of biological motion.

Johansson filmed walkers and runners in a dark room with lights attached to their main joints and demonstrated that such moving light spots were perceived as human movements. To extend this finding the detection and recognition of Johansson displays of different kinds of movements under three light-spot conditions were studied to determine how human actions are perceived on the basis of biological-motion information. Locomotory, instrumental, and social actions were presented in each condition, namely in normal Johansson (light attached to joints), inter-joint (light attached between joints), and upside-down Johansson. Subjects' verbal responses and recognition times were measured. Locomotory actions were recognised better and faster than social and instrumental actions. Furthermore, biological motions were recognised much better and faster when the light-spot displays were presented in the normal orientation rather than upside down. Recognition rate was only slightly impaired under the inter-joint condition. It is argued that the perceptual analysis of actions and movements starts primarily on an intermediate level of action coding and comprises more than just the similarity of movement patterns or simple structures. Additionally, coding of dynamic phase relations and semantic coding take place at very early stages of the processing of biological motion. Implications of these results for computer vision, perceptual models, and mental representations are discussed.