Even in the absence of explicit stimulation, rats emit patterns of rhythmic whisking movements. Because of their stereotyped nature and their persistence after sensory denervation and cortical ablation, whisking movements have been assumed to reflect the output of a central pattern generator (CPG). However, identification of a movement pattern as the product of a CPG requires evidence that its generation, patterning, and coordination are independent of sensory input. To provide such evidence, we used optoelectronic instrumentation to obtain high-resolution records of the movement trajectories of individual whiskers in rats whose heads were fixed to isolate their exploratory whisking from exafferent inputs. Unconditioned whisking patterns were quantitatively characterized by a biometric analysis of the kinematics, rhythmicity, and coordination of bilaterally homologous vibrissa movements. Unilateral and bilateral sectioning of the infraorbital nerve, which innervates the whiskers, was then performed to block reafferent inputs generated by the animal's own whisking movements. Unilateral sectioning of the nerve has no effect on whisking kinematics but is followed by a significant but relatively transient bilateral increase in whisking frequency. However, bilateral deafferentation, when performed in a single-stage procedure, does not disrupt the generation, patterning, or bilateral coordination of whisking patterns in the rat. These findings provide strong behavioral evidence for a whisking CPG and are discussed in relation to its possible location and properties.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a multimodular (domains A, B, C, D, E, and F) nuclear protein that participates in many fundamental cellular activities. Stimulated by binding to nicked DNA, PARP-1 catalyzes poly(ADP-ribosyl)ation of the acceptor proteins and itself using NAD(+) as a substrate. Early studies suggested that domain D is likely an interface for protein-protein interaction between PARP-1 and its targets and is also the primary region for automodification. However, determination of the modification sites has been complicated by the heterogeneous nature of the poly(ADP-ribose) polymer. Here we report a strategy to identify the modification sites on domain D using the PARP-1 E988Q mutant, which only catalyzes mono(ADP-ribosyl)ation. Trypsin digestion of the modified domain D followed by LC-MS/MS analysis led to the identification of three ADP-ribosylation sites in domain D (D387, E488, and E491). Our data also show, in contrast to early reports, that automodification of PARP-1 is not limited to domain D but occurs beyond this region. In addition, domain D is not essential for PARP-1 activity since PARP-1 mutant having domain D deleted is still catalytically active. Two synthetic peptides with amino acid sequences derived from the ADP-ribosylation sites of domain D were also demonstrated to act as PARP-1 substrates. The methodology and the results reported herein will facilitate future studies of PARP-1 catalysis.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a multimodular nuclear protein that participates in many fundamental cellular activities. Stimulated by binding to nicked DNA, PARP-1 catalyzes poly(ADP-ribosyl)ation of the acceptor proteins using NAD (+) as a substrate. In this work, NMR methods were used to determine the solution structure of human PARP-1 protein. Domain C was found to contain a zinc-binding motif of three antiparallel beta-strands with four conserved cysteines positioned to coordinate the metal ligand, in addition to a helical region. The zinc-binding motif is structurally reminiscent of the "zinc-ribbon" fold, but with a novel spacing between the conserved cysteines (CX2CX12CX 9C). Domain C alone does not appear to bind to DNA. Interestingly, domain C is essential for PARP-1 activity, since a mixture containing nicked DNA and the PARP-1 ABDEF domains has only basal enzymatic activity, while the addition of domain C to the mixture initiated NAD (+) hydrolysis and the formation of poly(ADP-ribose), as detected by an NMR-based assay and autoradiography. The structural model for domain C in solution provides an important framework for further studies aimed at improving our understanding of how the various domains within the complex PARP-1 enzyme play their respective roles in regulating the enzyme activity when cells are under conditions of genotoxic stress.

The rat's mystacial vibrissae are active during exploratory and discriminative behaviors, with individual vibrissae serving as elements in a receptive array scanned across object surfaces. To facilitate neurobehavioral analysis of this sensorimotor system, we have developed an experimental paradigm that confines vibrissa movements to a defined physical location, makes possible on-line monitoring of "whisking" activity, and brings such activity under associative control using operant conditioning procedures. Rats were secured, and movements of an identified bilaterally homologous pair of vibrissae (right and left gamma straddlers) were detected by laser-based photodetectors. Subjects were maintained on a water deprivation schedule, and whisker movements were monitored during adaptation to the test situation and after the clipping of other vibrissae on both sides of the snout. Rats were reinforced with water delivery for emitting vibrissa movements in the presence of a conditioned stimulus (tone) whose presentation was made contingent upon a prior period of nonwhisking. The rate and temporal distribution of vibrissa movements were brought under experimental control by means of interval and ratio reinforcement schedules. Although the procedures provide minimal information about the kinematics or topography of conditioned vibrissa movements, they permit the investigator to manipulate response parameters normally under the voluntary control of the animal in a preparation amenable to neurophysiological analysis.

Previous studies, based on qualitative observations, reported that lesions of the whisker motor cortex produce no deficits in whisking behavior. We used high-resolution optoelectronic recording methods to compare the temporal organization and kinematics of whisker movements before and after unilateral lesions of whisker motor cortex in rats. We now report that while the lesion did not abolish whisking, it significantly disrupted whisking kinematics, coordination, and temporal organization. Lesioned animals showed significant increases in the velocity and amplitude of whisker protractions contralateral to the lesions, as well as a reduction in the synchrony of whisker movements on the two sides of the face. There was a marked shift in the distribution of whisking frequencies, with reduction of activity in the 5-7 Hz bandwidth and increased activity at < 2 Hz. Disruptions of the normal whisking pattern were evident on both sides of the face, and the magnitude of these effects was proportional to the extent of the cortical ablation. We suggest that the observed deficits reflect an imbalance in cortical inputs to a brainstem central pattern generator.

Long-term memory (LTM) formation requires new protein synthesis and new gene expression. Based on our work in Aplysia, we hypothesized that the rRNA genes, stimulation-dependent targets of the enzyme Poly(ADP-ribose) polymerase-1 (PARP-1), are primary effectors of the activity-dependent changes in synaptic function that maintain synaptic plasticity and memory. Using electrophysiology, immunohistochemistry, pharmacology and molecular biology techniques, we show here, for the first time, that the maintenance of forskolin-induced late-phase long-term potentiation (L-LTP) in mouse hippocampal slices requires nucleolar integrity and the expression of new rRNAs. The activity-dependent upregulation of rRNA, as well as L-LTP expression, are poly(ADP-ribosyl)ation (PAR) dependent and accompanied by an increase in nuclear PARP-1 and Poly(ADP) ribose molecules (pADPr) after forskolin stimulation. The upregulation of PARP-1 and pADPr is regulated by Protein kinase A (PKA) and extracellular signal-regulated kinase (ERK)—two kinases strongly associated with long-term plasticity and learning and memory. Selective inhibition of RNA Polymerase I (Pol I), responsible for the synthesis of precursor rRNA, results in the segmentation of nucleoli, the exclusion of PARP-1 from functional nucleolar compartments and disrupted L-LTP maintenance. Taken as a whole, these results suggest that new rRNAs (28S, 18S, and 5.8S ribosomal components)—hence, new ribosomes and nucleoli integrity—are required for the maintenance of long-term synaptic plasticity. This provides a mechanistic link between stimulation-dependent gene expression and the new protein synthesis known to be required for memory consolidation.

Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

Cities play an important role in fostering and amplifying the transmission of airborne diseases (e.g., influenza) because of dense human contacts. Before an outbreak of airborne diseases within a city, how to determine an appropriate containment area for effective vaccination strategies is unknown. This research treats airborne disease spreads as geo-social interaction patterns, because viruses transmit among different groups of people over geographical locations through human interactions and population movement. Previous research argued that an appropriate scale identified through human geo-social interaction patterns can provide great potential for effective vaccination. However, little work has been done to examine the effectiveness of such vaccination at large scales (e.g., city) that are characterized by spatially heterogeneous population distribution and movement. This article therefore aims to understand the impact of geo-social interaction patterns on effective vaccination in the urbanized area of Portland, Oregon. To achieve this goal, we simulate influenza transmission on a large-scale location-based social network to 1) identify human geo-social interaction patterns for designing effective vaccination strategies, and 2) and evaluate the efficacy of different vaccination strategies according to the identified geo-social patterns. The simulation results illustrate the effectiveness of vaccination strategies based on geosocial interaction patterns in containing the epidemic outbreak at the source. This research can provide evidence to inform public health approaches to determine effective scales in the design of disease control strategies.