Cowpea mosaic virus: from the presentation of antigenic peptides to the display of active biomaterials.

The potential of cowpea mosaic virus (CPMV), a plant icosahedral virus, for the presentation of foreign peptides and proteins is reported. The most prominent feature at the virus surface is a region of the smaller of the two coat proteins (S) which has been extensively used for the insertion of foreign peptides. Given the availability of the three-dimensional structure of the native virus and the amenability of foreign peptide-expressing CPMV chimeras to crystallisation, immunological data can be correlated with the conformational state of the foreign insert. The latter is influenced by proteolysis which occurs within the foreign inserts. In an effort to offer an alternative context for peptide expression, extensive exploration of a second region of the S protein is reported with respect to tolerance to small insertions. Moreover, to make CPMV suitable for a wider spectrum of presentation, a technique was developed to allow surface coupling of a peptide which can serve as the anchoring point for a range of proteins. This new approach is also widely applicable for the direct chemical cross-linking of peptides and full-length protein domains to the viral capsid.

Folding of intracellular retinol and retinoic acid binding proteins.

The folding mechanisms of cellular retinol binding protein II (CRBP II), cellular retinoic acid binding protein I (CRABP I), and cellular retinoic acid binding protein II (CRABP II) were examined. These beta-sheet proteins have very similar structures and higher sequence homologies than most proteins in this diverse family. They have similar stabilities and show completely reversible folding at equilibrium with urea as a denaturant. The unfolding kinetics of these proteins were monitored during folding and unfolding by circular dichroism (CD) and fluorescence. During unfolding, CRABP II showed no intermediates, CRABP I had an intermediate with nativelike secondary structure, and CRBP II had an intermediate that lacked secondary structure. The refolding kinetics of these proteins were more similar. Each protein showed a burst-phase change in intensity by both CD and fluorescence, followed by a single observed phase by both CD and fluorescence and one or two additional refolding phases by fluorescence. The fluorescence spectral properties of the intermediate states were similar and suggested a gradual increase in the amount of native tertiary structure present for each step in a sequential path. However, the rates of folding differed by as much as 3 orders of magnitude and were slower than those expected from the contact order and topology of these proteins. As such, proteins with the same final structure may not follow the same route to the native state.

Folding mechanism of three structurally similar beta-sheet proteins.

The folding mechanism of cellular retinoic acid binding protein I (CRABP I), cellular retinol binding protein II (CRBP II), and intestinal fatty acid binding protein (IFABP) were investigated to determine if proteins with similar native structures have similar folding mechanisms. These mostly beta-sheet proteins have very similar structures, despite having as little as 33% sequence similarity. The reversible urea denaturation of these proteins was characterized at equilibrium by circular dichroism and fluorescence. The data were best fit by a two-state model for each of these proteins, suggesting that no significant population of folding intermediates were present at equilibrium. The native states were of similar stability with free energies (linearly extrapolated to 0 M urea, deltaGH2O) of 6.5, 8.3, and 5.5 kcal/mole for CRABP I, CRBP II, and IFABP, respectively. The kinetics of the folding and unfolding processes for these proteins was monitored by stopped-flow CD and fluorescence. Intermediates were observed during both the folding and unfolding of all of these proteins. However, the overall rates of folding and unfolding differed by nearly three orders of magnitude. Further, the spectroscopic properties of the intermediate states were different for each protein, suggesting that different amounts of secondary and/or tertiary structure were associated with each intermediate state for each protein. These data show that the folding path for proteins in the same structural family can be quite different, and provide evidence for different folding landscapes for these sequences.

Racial differences of frail elders in assistive technology.

This study compared differences in coping strategies, such as the use and ownership of assistive devices and home modifications, for 64 black and 441 white frail elders from the University at Buffalo Consumer Assessment Study. Controlling age, income, and education, there was no difference between black and white elders on health and functional status. Black elders on average own and use fewer assistive devices than white elders. There is no difference between the two groups in the number of home modifications, although black elders experience more home environmental problems than white elders. The majority of black elders rent their residence and thus lack the authority and ability to make the necessary adjustments to their home environmental problems. Predictor variables for assistive device use and environmental problems varied for black and white elders.

Sleep and purposive behavior: inverse deviations from randomness of neuronal firing patterns in the feline thalamus. A new form of homeostasis?

In behaving cats trained to press a bar for small aliquots of milk reward, single neuronal firing patterns were monitored from the nucleus reticularis (NR) thalami during bar bressing (BP), subsequent quiet wakefulness with EEG spindles (S- QW ), grooming behavior (GR) and slow-wave sleep (SWS). The temporal patterns in the neuronal spike trains were analyzed using a non-parametric method based on relative relations between sequential spike intervals. The deviations of pattern occurrences from the random model were quantified. During BP, specific patterns occurred much more often while others occurred much less often than predicted by the random model. Patterns that were dominant during BP, were selectively suppressed or virtually eliminated during S- QW , GR and SWS, despite the increased firing rate; and, vice versa, patterns that were suppressed below chance level during BP, became dominant during S- QW , GR and SWS. The magnitudes of these inversions of the statistical distribution of patterns were not random but graded and positively correlated, thus indicating that they were homeostatically controlled. Since the inversions were already evident shortly after the satiated ceased bar pressing, they may be related to the 'need' for sleep. On the basis of the known mechanisms of pattern generation and changes in receptors for putative transmitters, it was postulated that the inversions of pattern distribution are related to the recuperative function of SWS, i.e. resensitization of receptors that had been desensitized during the animal's stereotypic BP performance. The NR and other neuronal ensembles seem to constitute an oscillatory system with two modes of reciprocal connectivities : one is supporting wakefulness and emission of specific firing patterns, and the other is incompatible with wakefulness and instead is associated with inversion of statistical distribution of firing patterns and recuperative function of SWS.

Sleep-wakefulness: inverse deviation from randomness of neuronal firing patterns in the feline thalamus. A new form of homeostasis?

During stereotypic goal-directed behavior, neurons in the feline nucleus reticularis thalami emitted specific temporal patterns, while other patterns occurred much less often than predicted by the random model. During subsequent slow wave sleep, the mean firing rate increased, but the patterns that were emitted during behavior were eliminated or suppressed far below chance level, while those that were previously suppressed became dominant.

Effects of serotonin and morphine on spontaneous and evoked firing of nociceptive neurons in the trigeminal spinal nucleus of rats.

Spontaneously firing neurons that were responsive to noxious face pinch or noxious heat were studied in the trigeminal spinal nucleus of the rat brain. These neurons responded with either an increase or decrease in firing rate. In these neurons serotonin (5-hydroxytryptamine; 5-HT) apparently acts through two mechanisms to attenuate the response to a noxious stimulus. One mechanism is mimicked by morphine; these two drugs block the response to the noxious stimuli without having a consistent effect on spontaneous firing. The effects of the two drugs were somewhat selective depending on the noxious stimulus used and the effect of the noxious stimulus; morphine and 5-HT were more effective in blocking the increase in firing rate evoked by the face pinch but 5-HT and morphine were more effective in blocking the decrease in firing rate evoked by the noxious heat stimulus. Interestingly, the direction of the response to a particular noxious stimulus frequently predicted whether or not both morphine and 5-HT would act on the same or different neurons. A second mechanism by which 5-HT, but not morphine, acted was to change the spontaneous firing in a direction opposite that evoked by the noxious stimulus. This type of effect apparently modulated the response to a noxious stimulus by changing the spontaneous firing rate such that a noxious stimulus had to be more intense before it could significantly alter the neuronal firing in the opposite direction. Morphine occasionally produced a change in firing pattern in neurons; this effect remains to be documented more extensively.

Visual attention and neuronal firing patterns in the feline pulvinar nucleus of thalamus.

In behaving cats, temporal patterns of neuronal firing were studied during slow wave sleep (SWS), motionless quiet wakefulness (QW) coupled with specific direction of the animal's attention, and during bar pressing performance (BP) for milk reward. The analysis was based on relative relations between sequential spike intervals. The strength of the method is based on the fact that the probabilities of occurrence of patterns are determined by the history of a spike train. During SWS, the neuronal firing modes closely followed the theoretical model of independent distribution of intervals, whereas during QW and BP specific for each neuron departures from the model, i.e., patterning was observed. Most importantly, in seven chronically studied neurons idiosyncratic patterns were related to direction of the animal's attention, and, very likely, to the visual forms the animals gazed at, because the patterns disappeared in the dark and during SWS without major changes in the mean firing rate. The replications of patterns upon recurrence of a particular direction of attention was proven statistically. The constancy and idiosyncrasy of these patterns were apparent even though the comparable episodes occurred several hours apart, and the animals slept and/or ate in between, and the distance, i.e., the retinal size of visual forms varied from one episode to another. On the basis of correlative evidence, it was argued that, compared to more abstract modes of information processing, the identification and quantification of patterns based on relative relations between intervals require the least amount of storage of intermediate results. Hence, these patterns are likely to represent a simple and phylogenetically old principle of communication between neurons. It was postulated that the idiosyncrasy and invariance of patterns may play a role in constancy of feature extraction and Gestalt perception.

Phasic loss of constraints in timing of neuronal spikes in the feline centrum medianum during EEG alpha-like activity.

In unrestrained cats, temporal patterns of single neuronal firing in the centrum medianum-parafascicular complex (CM-Pf) of thalamus were studied during a state of motionless quiet wakefulness. The spike trains from each neuron were electronically divided into episodes that occurred during desynchronized EEG and those that occurred during bursts of 6-14 Hz EEG spindles or alpha-like activity over the parieto-occipital cortex and in the CM-Pf. Contrary to expectations based on the theory of inhibitory phasing of neuronal activity, the episodes of synchronized quiet wakefulness (S-QW) were associated with independent and random distribution of spike intervals, although they tended to occur in clusters. During episodes of desynchronized quiet wakefulness (D-QW), significant temporal patterns were emitted by most neurons studied. The results suggest that: (a) during D-QW and increased levels of vigilance, temporal patterns are generated by cognitive processes and enhanced specific connectivities between CM-Pf neurons and other systems; (b) if connectivity is defined as increased certainty of synaptic transmission and iterative activation of the same pathways, then the assumption that the gross EEG thalamo-cortical synchronization represents increased connectivities between the CM-Pf neurons and other systems, may be erroneous; and (c) since temporal patterns of single neuronal discharges are determined by specific spatio-temporal distribution of synaptic drive, therefore during EEG spindles, most EPSP-IPSP sequences impinging upon CM-Pf neurons are conveyed by randomly varying polysynaptic pathways and have random spatio-temporal distribution on the soma and dendrites. In light of other observations, such a process is equivalent to an active introduction of uncertainty or 'entropy' into the information processing system, a state which may be important in preserving plasticity of operational modes of CM-Pf neurons and those with which they directly and/or indirectly interact.

Neuronal firing patterns in the feline hippocampus during sleep and wakefulness.

The study addressed the problem of information transmission in mammalian brain as reflected in the emergence or disappearance of temporal patterns in extracellularly monitored single action potentials from the dorsal hippocampus of unrestrained cats during slow wave sleep (SWS), rapid eye movement sleep (REM), and motionless quiet wakefulness (QW). The spike trains were analyzed with a nonparametric technique. Chi-square statistics were used to measure deviation of firing patterns from the theoretical model which is based on the assumption that the intervals are random and/or independent from each other. The plots of the chi-square values for a given set of patterns represented the neuronal 'signatures' characteristic of a behavioral state. During SWS most neurons followed the theoretical model, i.e. their 'signatures' were flat and statistically non-significant. However, during REM sleep and QW their firing modes showed specific deviations from the theoretical model: some patterns occurred more often while others less often than expected, thus generating large and statistically significant 'signatures'. During REM sleep some neurons shared similar tendencies in their departures from the theoretical model. However, during QW the same neurons developed their individual 'signatures' which were significantly different from each other. Hence, the QW episodes were characterized by a greater differentiation of neuronal firing patterns. The mean firing rate and the shape of the time interval histogram were not necessarily correlated with the emergence of specific temporal patterns in spike trains. The results suggest that information transmission from one neuron to another depends on the emergence of repetitive and specific temporal patterns. The strong tendency of most neurons to lapse during SWS into a firing mode that closely follows the theoretical model constitutes the basis for a working hypothesis which states that the essence of SWS recovery in cognitive systems is the disappearance of temporal patterns, and that the 'noisy' interactions between neurons plays an important role in the recuperative processes.