AMPA receptor translocation and phosphorylation are induced by transcranial direct current stimulation in rats.

Abstract Over the last decade, the interest in transcranial direct current stimulation (tDCS) has continued to increase, along with consideration of how it affects neuroplasticity mechanisms in the brain. Both human and animal studies have demonstrated numerous benefits and, although its application has increased, the neurophysiological mechanisms underlying tDCS' beneficial effects remain largely unknown. Recent studies have shown that long-term potentiation (LTP) increases following tDCS. In this work, we utilized a rodent model of tDCS to directly assess changes in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, a critical protein for enhancing synaptic transmission. Animals were subjected to 250 μA of direct current (DC) stimulation for 30 min with immediate tissue collection. Translocation and phosphorylation of AMPA receptors were examined using protein immunoblot analysis following a subcellular fractionation method. Our findings show that a single application of in vivo tDCS can affect both the translocation and phosphorylation of AMPA receptors in the hippocampus while increasing AMPA receptor phosphorylation in the hypothalamus. In the hippocampus, tDCS increased AMPA translocation to the synapse and increased the phosphorylation of the S831 site on GluA1. In the hypothalamus, no statistically significant changes were observed in AMPA translocation while an increase in the phosphorylation of the S831 site was observed. No changes in the phosphorylation of GluA1 at the S845 site were detected in either brain region. In sum, our findings identify specific AMPA receptor changes induced by tDCS, thereby providing further details on the mechanisms by which tDCS could affect the establishment of LTP and modulate neuroplasticity

Investigating the biochemistry of phenylpropanoid metabolism in basil and petunia

Land plants are, in general, immobile organisms. This sedentary lifestyle leaves them vulnerable to a wide variety of adversity, thus an even wider variety of adaptations must be made to ensure successful reproduction. Plant secondary metabolites, of which more than 100,000 structures have been described to date, represent plant life\u27s attempt to cope with demanding environments encountered globally. Volatile secondary metabolites enable plants to boost reproductive efficiency and genetic diversity through attraction of pollinators, provide constitutive and inducible defenses against attack from herbivores and pathogens, mitigate abiotic stresses encountered in nature, increase species’ distribution via enticement of seed dispersers, and even warn neighboring plants of danger from pests and pathogens. Working in basil and petunia, my research has characterized two critical steps in secondary metabolism. Many basil cultivars as well as other plant taxa produce the volatile compound methyl cinnamate. In basil, this compound is produced via transfer of a reactive methyl group from S-adenosyl methionine to the carboxyl group of trans-cinnamic acid, a reaction catalyzed by cinnamate/p-coumarate carboxylmethyltransferase (CCMT) enzymes. These enzymes are localized within the pelatate glandular trichomes of basil leaves and have somewhat broad substrate specificity towards small carboxylic acids. Amino acid residues determining substrate specificity of these enzymes were probed using site directed mutagenesis and novel activity with additional carboxylic acids was engineered in a mutant form of one CCMT isoform. In petunia, the phenylpropene isoeugenol makes up a significant portion of emitted volatile compounds from floral tissues. Recent work described formation of isoeugenol and its positional isomer eugenol from the compound coniferyl acetate however the reaction forming coniferyl acetate from its precursor coniferyl alcohol was undescribed. Investigation of this step included isolation of the candidate gene, gene expression profiling, and biochemical characterization of the recombinant enzyme

Evolution of Cinnamate/p-Coumarate Carboxyl Methyltransferases and Their Role in the Biosynthesis of Methylcinnamate[W]

Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil ( Ocimum basilicum ) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p -coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/ p -coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase–like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings

Evaluation of thermal desorption analysis on a portable GC–MS system

<p>The HAPSITE-ER-TD (Hazardous Air Pollutants on Site Extended Range HAPSITE-ER) portable gas chromatograph–mass spectrometer (GC–MS) combines the sensitivity of a thermal desorption (TD) GC–MS with the advantages of field-portable instrumentation. Though previous iterations of the HAPSITE have been extensively evaluated in the literature, performance assessment of the TD-equipped instrument is lacking. In this manuscript, the variability in the HAPSITE-ER-TD response was established for both internal standards and test compounds across three instruments over a 5-week time course. These data show poor normalised internal standard reproducibility with %RSD values from 17.84% to 49.97% on the HAPSITE-ER-TD when compared to a bench-top instrument (%RSD < 11.6%), suggesting that use of TD tubes preloaded with an internal standard may be valuable for normalisation purposes. Though our determined method detection limit (MDL) values reveal that substantial variabilities exist between separate HAPSITE-ER-TD systems, MDL values comparable to the standard bench-top equipment can be achieved. Additionally, data generated with the TO-15/TO-17 65 component target compound mix and JP-8 jet fuel show statistically significant (<i>p</i> value = 0.0014) compound-dependent system carryover on the HAPSITE-ER-TD, indicating that procedural modifications to eliminate instrumental carryover may be necessary. This study establishes several limitations associated with the use of the HAPSITE-ER TD accessory with suggestions for addressing the shortcomings to allow for reliable field use.</p

Single-molecule stretching studies of RNA chaperones

RNA chaperone proteins play significant roles in diverse biological contexts. The most widely studied RNA chaperones are the retroviral nucleocapsid proteins (NC), also referred to as nucleic acid (NA) chaperones. Surprisingly, the biophysical properties of the NC proteins vary significantly for different viruses, and it appears that HIV-1 NC has optimal NA chaperone activity. In this review we discuss the physical nature of the NA chaperone activity of NC. We conclude that the optimal NA chaperone must saturate NA binding, leading to strong NA aggregation and slight destabilization of all NA duplexes. Finally, rapid kinetics of the chaperone protein interaction with NA is another primary component of its NA chaperone activity. We discuss these characteristics of HIV-1 NC and compare them with those of other NA binding proteins and ligands that exhibit only some characteristics of NA chaperone activity, as studied by single molecule DNA stretching

Features, processing states and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function

Nucleocapsid (NC) is central to retroviral replication. Nucleic acid chaperoning is a key function for NC through the action of its conserved basic amino acids and zinc-finger structures. NC manipulates genomic RNA from its packaging in the producer cell to reverse transcription into the infected host cell. This chaperone function, in conjunction with NCs aggregating properties, is up-modulated by successive NC processing events, from the Gag precursor to the fully mature protein, resulting in the condensation of the nucleocapsid within the capsid shell. Reverse transcription also depends on NC processing, whereas this process provokes NC dissociation from double-stranded DNA, leading to a preintegration complex (PIC), competent for host chromosomal integration. In addition NC interacts with cellular proteins, some of which are involved in viral budding, and also with several viral proteins. All of these properties are reviewed here, focusing on HIV-1 as a paradigmatic reference and highlighting the plasticity of the nucleocapsid architecture