Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control

Numerous studies have implicated the pRB family of nuclear proteins in the control of cell cycle progression. Although over-expression experiments have revealed that each of these proteins, pRB, p107, and p130, can induce a G1 cell cycle arrest, mouse knockouts demonstrated distinct developmental requirements for these proteins, as well as partial functional redundancy between family members. To study the mechanism by which the closely related pRB family proteins contribute to cell cycle progression, we generated 3T3 fibroblasts derived from embryos that lack one or more of these proteins (pRB−/−, p107−/−, p130−/−, pRB−/−/p107−/−, pRB−/−/p130−/−, and p107−/−/p130−/−). By comparing the growth and cell cycle characteristics of these cells, we have observed clear differences in the manner in which they transit through the G1 and S phases as well as exit from the cell cycle. Deletion of Rb, or more than one of the family members, results in a shortening of G1 and a lengthening of S phase, as well as a reduction in growth factor requirements. In addition, the individual cell lines showed differential regulation of a subset of E2F-dependent gene promoters, as well as differences in cell cycle-dependent kinase activity. Taken together, these observations suggest that the closely related pRB family proteins affect cell cycle progression through distinct biochemical mechanisms and that their coordinated action may contribute to their diverse functions in various physiological settings.

Disfacilitation and active inhibition in the neocortex during the natural sleep-wake cycle: An intracellular study

Earlier extracellular recordings during natural sleep have shown that, during slow-wave sleep (SWS), neocortical neurons display long-lasting periods of silence, whereas they are tonically active and discharge at higher rates during waking and sleep with rapid eye movements (REMs). We analyzed the nature of long-lasting periods of neuronal silence in SWS and the changes in firing rates related to ocular movements during REM sleep and waking using intracellular recordings from electrophysiologically identified neocortical neurons in nonanesthetized and nonparalyzed cats. We found that the silent periods during SWS are associated with neuronal hyperpolarizations, which are due to a mixture of K+ currents and disfacilitation processes. Conventional fast-spiking neurons (presumably local inhibitory interneurons) increased their firing rates during REMs and eye movements in waking. During REMs, the firing rates of regular-spiking neurons from associative areas decreased and intracellular traces revealed numerous, short-lasting, low-amplitude inhibitory postsynaptic potentials (IPSPs), that were reversed after intracellular chloride infusion. In awake cats, regular-spiking neurons could either increase or decrease their firing rates during eye movements. The short-lasting IPSPs associated with eye movements were still present in waking; they preceded the spikes and affected their timing. We propose that there are two different forms of firing rate control: disfacilitation induces long-lasting periods of silence that occur spontaneously during SWS, whereas active inhibition, consisting of low-amplitude, short-lasting IPSPs, is prevalent during REMs and precisely controls the timing of action potentials in waking.

Formation of temporal-feature maps by axonal propagation of synaptic learning

Computational maps are of central importance to a neuronal representation of the outside world. In a map, neighboring neurons respond to similar sensory features. A well studied example is the computational map of interaural time differences (ITDs), which is essential to sound localization in a variety of species and allows resolution of ITDs of the order of 10 μs. Nevertheless, it is unclear how such an orderly representation of temporal features arises. We address this problem by modeling the ontogenetic development of an ITD map in the laminar nucleus of the barn owl. We show how the owl's ITD map can emerge from a combined action of homosynaptic spike-based Hebbian learning and its propagation along the presynaptic axon. In spike-based Hebbian learning, synaptic strengths are modified according to the timing of pre- and postsynaptic action potentials. In unspecific axonal learning, a synapse's modification gives rise to a factor that propagates along the presynaptic axon and affects the properties of synapses at neighboring neurons. Our results indicate that both Hebbian learning and its presynaptic propagation are necessary for map formation in the laminar nucleus, but the latter can be orders of magnitude weaker than the former. We argue that the algorithm is important for the formation of computational maps, when, in particular, time plays a key role.

Cue recognition and cue elaboration in learning from examples.

This paper describes the processes used by students to learn from worked-out examples and by working through problems. Evidence is derived from protocols of students learning secondary school mathematics and physics. The students acquired knowledge from the examples in the form of productions (condition-->action): first discovering conditions under which the actions are appropriate and then elaborating the conditions to enhance efficiency. Students devoted most of their attention to the condition side of the productions. Subsequently, they generalized the productions for broader application and acquired specialized productions for special problem classes.

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Phenylalanine ammonia-lyase (EC 4.3.1.5) from parsley is posttranslationally modified by dehydrating its Ser-202 to the catalytically essential dehydroalanine prosthetic group. The codon of Ser-202 was changed to those of alanine and threonine by site-directed mutagenesis. These mutants and the recombinant wild-type enzyme, after treatment with sodium borohydride, were virtually inactive with L-phenylalanine as substrate but catalyzed the deamination of L-4-nitrophenylalanine, which is also a substrate for the wild-type enzyme. Although the mutants reacted about 20 times slower with L-4-nitrophenylalanine than the wild-type enzyme, their Vmax for L-4-nitrophenylalanine was two orders of magnitude higher than for L-phenylalanine. In contrast to L-tyrosine, which was a poor substrate, DL-3-hydroxyphenylalanine (DL-m-tyrosine) was converted by phenylalanine ammonia-lyase at a rate comparable to that of L-phenylalanine. These results suggest a mechanism in which the crucial step is an electrophilic attack of the prosthetic group at position 2 or 6 of the phenyl group. In the resulting carbenium ion, the beta-HSi atom is activated in a similar way as it is in the nitro analogue. Subsequent elimination of ammonia, concomitant with restoration of both the aromatic ring and the prosthetic group, completes the catalytic cycle.