Engineering precision RNA molecular switches

Ligand-specific molecular switches composed of RNA were created by coupling preexisting catalytic and receptor domains via structural bridges. Binding of ligand to the receptor triggers a conformational change within the bridge, and this structural reorganization dictates the activity of the adjoining ribozyme. The modular nature of these tripartite constructs makes possible the rapid construction of precision RNA molecular switches that trigger only in the presence of their corresponding ligand. By using similar enzyme engineering strategies, new RNA switches can be made to operate as designer molecular sensors or as a new class of genetic control elements.

Targeting Gβγ signaling in arterial vascular smooth muscle proliferation: A novel strategy to limit restenosis

Restenosis continues to be a major problem limiting the effectiveness of revascularization procedures. To date, the roles of heterotrimeric G proteins in the triggering of pathological vascular smooth muscle (VSM) cell proliferation have not been elucidated. βγ subunits of heterotrimeric G proteins (Gβγ) are known to activate mitogen-activated protein (MAP) kinases after stimulation of certain G protein-coupled receptors; however, their relevance in VSM mitogenesis in vitro or in vivo is not known. Using adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of Gβγ signaling (βARKct), we evaluated the role of Gβγ in MAP kinase activation and proliferation in response to several mitogens, including serum, in cultured rat VSM cells. Our results include the striking finding that serum-induced proliferation of VSM cells in vitro is mediated largely via Gβγ. Furthermore, we studied the effects of in vivo adenoviral-mediated βARKct gene transfer on VSM intimal hyperplasia in a rat carotid artery restenosis model. Our in vivo results demonstrated that the presence of the βARKct in injured rat carotid arteries significantly reduced VSM intimal hyperplasia by 70%. Thus, Gβγ plays a critical role in physiological VSM proliferation, and targeted Gβγ inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.

Submicrosecond pacemaker precision is behaviorally modulated: The gymnotiform electromotor pathway

What are the limits and modulators of neural precision? We address this question in the most regular biological oscillator known, the electric organ command nucleus in the brainstem of wave-type gymnotiform fish. These fish produce an oscillating electric field, the electric organ discharge (EOD), used in electrolocation and communication. We show here that the EOD precision, measured by the coefficient of variation (CV = SD/mean period) is as low as 2 × 10−4 in five species representing three families that range widely in species and individual mean EOD frequencies (70–1,250 Hz). Intracellular recording in the pacemaker nucleus (Pn), which commands the EOD cycle by cycle, revealed that individual Pn neurons of the same species also display an extremely low CV (CV = 6 × 10−4, 0.8 μs SD). Although the EOD CV can remain at its minimum for hours, it varies with novel environmental conditions, during communication, and spontaneously. Spontaneous changes occur as abrupt steps (250 ms), oscillations (3–5 Hz), or slow ramps (10–30 s). Several findings suggest that these changes are under active control and depend on behavioral state: mean EOD frequency and CV can change independently; CV often decreases in response to behavioral stimuli; and lesions of one of the two inputs to the Pn had more influence on CV than lesions of the other input.

Interleaved concatenated codes: New perspectives on approaching the Shannon limit

The last few years have witnessed a significant decrease in the gap between the Shannon channel capacity limit and what is practically achievable. Progress has resulted from novel extensions of previously known coding techniques involving interleaved concatenated codes. A considerable body of simulation results is now available, supported by an important but limited theoretical basis. This paper presents a computational technique which further ties simulation results to the known theory and reveals a considerable reduction in the complexity required to approach the Shannon limit.

High-precision 40Ar/39Ar geochronology and the advent of North America’s Late Cretaceous terrestrial fauna

A densely sampled, diverse new fauna from the uppermost Cedar Mountain Formation, Utah, indicates that the basic pattern of faunal composition for the Late Cretaceous of North America was already established by the Albian-Cenomanian boundary. Multiple, concordant 40Ar/39Ar determinations from a volcanic ash associated with the fauna have an average age of 98.39 ± 0.07 million years. The fauna of the Cedar Mountain Formation records the first global appearance of hadrosaurid dinosaurs, advanced lizard (e.g., Helodermatidae), and mammal (e.g., Marsupialia) groups, and the first North American appearance of other taxa such as tyrannosaurids, pachycephalosaurs, and snakes. Although the origin of many groups is unclear, combined biostratigraphic and phylogenetic evidence suggests an Old World, specifically Asian, origin for some of the taxa, an hypothesis that is consistent with existing evidence from tectonics and marine invertebrates. Large-bodied herbivores are mainly represented by low-level browsers, ornithopod dinosaurs, whose radiations have been hypothesized to be related to the initial diversification of angiosperm plants. Diversity at the largest body sizes (>106 g) is low, in contrast to both preceding and succeeding faunas; sauropods, which underwent demise in the Northern hemisphere coincident with the radiation of angiosperms, apparently went temporarily unreplaced by other megaherbivores. Morphologic and taxonomic diversity among small, omnivorous mammals, multituberculates, is also low. A later apparent increase in diversity occurred during the Campanian, coincident with the appearance of major fruit types among angiosperms, suggesting the possibility of adaptive response to new resources.

Minimum size limit for useful locomotion by free-swimming microbes

Formulas are derived for the effect of size on a free-swimming microbe’s ability to follow chemical, light, or temperature stimuli or to disperse in random directions. The four main assumptions are as follows: (i) the organisms can be modeled as spheres, (ii) the power available to the organism for swimming is proportional to its volume, (iii) the noise in measuring a signal limits determination of the direction of a stimulus, and (iv) the time available to determine stimulus direction or to swim a straight path is limited by rotational diffusion caused by Brownian motion. In all cases, it is found that there is a sharp size limit below which locomotion has no apparent benefit. This size limit is estimated to most probably be about 0.6 μm diameter and is relatively insensitive to assumed values of the other parameters. A review of existing descriptions of free-floating bacteria reveals that the smallest of 97 motile genera has a mean length of 0.8 μm, whereas 18 of 94 nonmotile genera are smaller. Similar calculations have led to the conclusion that a minimum size also exists for use of pheromones in mate location, although this size limit is about three orders of magnitude larger. In both cases, the application of well-established physical laws and biological generalities has demonstrated that a common feature of animal behavior is of no use to small free-swimming organisms.

The structure and precision of retinal spike trains

Assessing the reliability of neuronal spike trains is fundamental to an understanding of the neural code. We measured the reproducibility of retinal responses to repeated visual stimuli. In both tiger salamander and rabbit, the retinal ganglion cells responded to random flicker with discrete, brief periods of firing. For any given cell, these firing events covered only a small fraction of the total stimulus time, often less than 5%. Firing events were very reproducible from trial to trial: the timing jitter of individual spikes was as low as 1 msec, and the standard deviation in spike count was often less than 0.5 spikes. Comparing the precision of spike timing to that of the spike count showed that the timing of a firing event conveyed several times more visual information than its spike count. This sparseness and precision were general characteristics of ganglion cell responses, maintained over the broad ensemble of stimulus waveforms produced by random flicker, and over a range of contrasts. Thus, the responses of retinal ganglion cells are not properly described by a firing probability that varies continuously with the stimulus. Instead, these neurons elicit discrete firing events that may be the fundamental coding symbols in retinal spike trains.

A Single Limit Dextrinase Gene Is Expressed Both in the Developing Endosperm and in Germinated Grains of Barley1

The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 3.2.1.41) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain.

Osmotic Water Permeability of Isolated Protoplasts. Modifications during Development1

A transference chamber was developed to measure the osmotic water permeability coefficient (Pos) in protoplasts 40 to 120 μm in diameter. The protoplast was held by a micropipette and submitted to a steep osmotic gradient created in the transference chamber. Pos was derived from the changes in protoplast dimensions, as measured using a light microscope. Permeabilities were in the range 1 to 1000 μm s−1 for the various types of protoplasts tested. The precision for Pos was ≤40%, and within this limit, no asymmetry in the water fluxes was observed. Measurements on protoplasts isolated from 2- to 5-d-old roots revealed a dramatic increase in Pos during root development. A shift in Pos from 10 to 500 μm s−1 occurred within less than 48 h. This phenomenon was found in maize (Zea mays), wheat (Triticum aestivum), and rape (Brassica napus) roots. These results show that early developmental processes modify water-transport properties of the plasma membrane, and that the transference chamber is adapted to the study of water-transport mechanisms in native membranes.

Glutamate antagonists limit tumor growth

Neuronal progenitors and tumor cells possess propensity to proliferate and to migrate. Glutamate regulates proliferation and migration of neurons during development, but it is not known whether it influences proliferation and migration of tumor cells. We demonstrate that glutamate antagonists inhibit proliferation of human tumor cells. Colon adenocarcinoma, astrocytoma, and breast and lung carcinoma cells were most sensitive to the antiproliferative effect of the N-methyl-d-aspartate antagonist dizocilpine, whereas breast and lung carcinoma, colon adenocarcinoma, and neuroblastoma cells responded most favorably to the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate antagonist GYKI52466. The antiproliferative effect of glutamate antagonists was Ca2+ dependent and resulted from decreased cell division and increased cell death. Morphological alterations induced by glutamate antagonists in tumor cells consisted of reduced membrane ruffling and pseudopodial protrusions. Furthermore, glutamate antagonists decreased motility and invasive growth of tumor cells. These findings suggest anticancer potential of glutamate antagonists.