The cyclic AMP cascade is altered in the fragile X nervous system

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.     

A Simple ERP Method for Quantitative Analysis of Cognitive Workload in Myoelectric Prosthesis Control and Human-Machine Interaction

Common goals in the development of human-machine interface (HMI) technology are to reduce cognitive workload and increase function. However, objective and quantitative outcome measures assessing cognitive workload have not been standardized for HMI research. The present study examines the efficacy of a simple event-related potential (ERP) measure of cortical effort during myoelectric control of a virtual limb for use as an outcome tool. Participants trained and tested on two methods of control, direct control (DC) and pattern recognition control (PRC), while electroencephalographic (EEG) activity was recorded. Eighteen healthy participants with intact limbs were tested using DC and PRC under three conditions: passive viewing, easy, and hard. Novel auditory probes were presented at random intervals during testing, and significant task-difficulty effects were observed in the P200, P300, and a late positive potential (LPP), supporting the efficacy of ERPs as a cognitive workload measure in HMI tasks. LPP amplitude distinguished DC from PRC in the hard condition with higher amplitude in PRC, consistent with lower cognitive workload in PRC relative to DC for complex movements. Participants completed trials faster in the easy condition using DC relative to PRC, but completed trials more slowly using DC relative to PRC in the hard condition. The results provide promising support for ERPs as an outcome measure for cognitive workload in HMI research such as prosthetics, exoskeletons, and other assistive devices, and can be used to evaluate and guide new technologies for more intuitive HMI control.     

Real-time Investigation of SV40 Large T-antigen Helicase Activity Using Surface Plasmon Resonance

The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.

Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.     

Amino acid variations resulting in functional and nonfunctional zebrafish P2X<Subscript>1</Subscript> and P2X<Subscript>5.1</Subscript> receptors

Several zebrafish P2X receptors (zP2X(1), zP2X(2), and zP2X(5.1)) have been reported to produce little or no current although their mammalian orthologs produce functional homomeric receptors. We isolated new cDNA clones for these P2X receptors that revealed sequence variations in each. The new variants of zP2X(1) and zP2X(5.1) produced substantial currents when expressed by Xenopus oocytes, however the new variant of zP2X(2) was still nonfunctional. zP2X(2) lacks two lysine residues essential for ATP responsiveness in other P2X receptors; however introduction of these two lysines was insufficient to allow this receptor to function as a homotrimer. We also tested whether P2X signaling is required for myogenesis or synaptic communication at the zebrafish neuromuscular junction. We found that embryonic skeletal muscle expressed only one P2X receptor, P2X(5.1). Antisense knockdown of P2X(5.1) eliminated skeletal muscle responsiveness to ATP but did not prevent myogenesis or behaviors that require functional transmission at the neuromuscular junction.     

Intrinsic regulation of spatiotemporal organization within the suprachiasmatic nucleus

The mammalian pacemaker in the suprachiasmatic nucleus (SCN) contains a population of neural oscillators capable of sustaining cell-autonomous rhythms in gene expression and electrical firing. A critical question for understanding pacemaker function is how SCN oscillators are organized into a coherent tissue capable of coordinating circadian rhythms in behavior and physiology. Here we undertake a comprehensive analysis of oscillatory function across the SCN of the adult PER2::LUC mouse by developing a novel approach involving multi-position bioluminescence imaging and unbiased computational analyses. We demonstrate that there is phase heterogeneity across all three dimensions of the SCN that is intrinsically regulated and extrinsically modulated by light in a region-specific manner. By investigating the mechanistic bases of SCN phase heterogeneity, we show for the first time that phase differences are not systematically related to regional differences in period, waveform, amplitude, or brightness. Furthermore, phase differences are not related to regional differences in the expression of arginine vasopressin and vasoactive intestinal polypeptide, two key neuropeptides characterizing functionally distinct subdivisions of the SCN. The consistency of SCN spatiotemporal organization across individuals and across planes of section suggests that the precise phasing of oscillators is a robust feature of the pacemaker important for its function.     

The diheme cytochrome c peroxidase from Shewanella oneidensis requires reductive activation

We report the characterization of the diheme cytochrome c peroxidase (CcP) from Shewanella oneidensis (So) using UV-visible absorbance, electron paramagnetic resonance spectroscopy, and Michaelis-Menten kinetics. While sequence alignment with other bacterial diheme cytochrome c peroxidases suggests that So CcP may be active in the as-isolated state, we find that So CcP requires reductive activation for full activity, similar to the case for the canonical Pseudomonas type of bacterial CcP enzyme. Peroxide turnover initiated with oxidized So CcP shows a distinct lag phase, which we interpret as reductive activation in situ. A simple kinetic model is sufficient to recapitulate the lag-phase behavior of the progress curves and separate the contributions of reductive activation and peroxide turnover. The rates of catalysis and activation differ between MBP fusion and tag-free So CcP and also depend on the identity of the electron donor. Combined with Michaelis-Menten analysis, these data suggest that So CcP can accommodate electron donor binding in several possible orientations and that the presence of the MBP tag affects the availability of certain binding sites. To further investigate the structural basis of reductive activation in So CcP, we introduced mutations into two different regions of the protein that have been suggested to be important for reductive activation in homologous bacterial CcPs. Mutations in a flexible loop region neighboring the low-potential heme significantly increased the activation rate, confirming the importance of flexible loop regions of the protein in converting the inactive, as-isolated enzyme into the activated form.     

An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore

Commonly used monomeric blue fluorescent proteins suffer from moderate brightness. The brightest of them, mTagBFP, has a notably low chemical stability over time. Prolonged incubation of mTagBFP leads to its transition from a blue fluorescent state with absorbance at 401 nm to a non-fluorescent state with absorbance at 330 nm. Here, we have determined the chemical structure of the degraded product of the blue mTagBFP-like chromophore. On the basis of mTagBFP we have developed an improved variant, named mTagBFP2. mTagBFP2 exhibits 2-fold greater chemical stability and substantially higher brightness in live cells than mTagBFP. mTagBFP2 is also 1.2-fold and 1.7-fold more photostable than mTagBFP in widefield and confocal microscopy setups, respectively. mTagBFP2 maintains all other beneficial properties of the parental mTagBFP including the high pH stability and fast chromophore formation. The enhanced photostability and chromophore chemical stability of mTagBFP2 make it a superior protein tag. mTagBFP2 performs well in the numerous protein fusions and surpasses mTagBFP as a donor in Förster resonance energy transfer with several green fluorescent protein acceptors.