Nicotine increases dopamine clearance in medial prefrontal cortex in rats raised in an enriched environment

Environmental enrichment results in differential behavioral and neurochemical responsiveness to nicotine. The present study investigates dopamine clearance (CL_DA) in striatum and medial prefrontal cortex (mPFC) using in vivo voltammetry in rats raised in enriched (EC) or impoverished conditions (IC) and administered nicotine (0.4 mg/kg) or saline. Baseline CL_DA in striatum or mPFC was not different between EC and IC. Across repeated DA application, striatal CL_DA increased in saline-control EC and IC. CL_DA increased in mPFC in saline-control IC; CL_DA did not change in saline-control EC. Thus, enrichment differentially alters dynamic responses of the dopamine transporter (DAT) to repeated DA application in mPFC, but not in striatum. In EC, nicotine increased mPFC CL_DA compared to saline-control, but had no effect on CL_DA in IC; nicotine had no effect in striatum in EC or IC. Compared to respective saline-controls, nicotine increased dihydroxyphenylacetic acid content in striatum and mPFC in EC, but not in IC. Nicotine also had no effect on DA content in striatum or mPFC in EC or IC. Results indicate that enrichment eliminated the dynamic response of mPFC DAT to repeated DA application in saline-control and augmented the nicotine-induced increase in DAT function in mPFC, but not in striatum.     

Actin polymerization upon processive capping by formin: a model for slowing and acceleration

Formin family proteins act as processive cappers of actin filaments, and determine the dynamics of a number of intracellular processes that are based on actin polymerization. The rate of filament growth upon processive capping varies within a broad range depending on the formin type and presence of profilin. While FH2 domains of various formins slow down polymerization by different extents, the FH1-FH2 domains in conjunction with profilin accelerate the reaction. Study of the physical mechanism of processive capping is vital for understanding the intracellular actin dynamics. We propose a model predicting that variation of a single physical parameter—the effective elastic energy of the formin-capped barbed end—results in the observed diversity of the polymerization rates. The model accounts for the whole range of the experimental results including the drastic slowing down of polymerization by FH2 of Cdc12 formin and the 4.5-fold acceleration of the reaction by FH1-FH2 of mDai1 formin in the presence of profilin. Fitting the theoretical predictions to the experimental curves provides the values of the effective elastic energies of different formin-barbed end complexes.     

Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase

Vaccinia virus DNA polymerase catalyzes duplex-by-duplex DNA joining reactions in vitro and many features of these recombination reactions are reprised in vivo. This can explain the intimate linkage between virus replication and genetic recombination. However, it is unclear why these apparently ordinary polymerases exhibit this unusual catalytic capacity. In this study, we have used different substrates to perform a detailed investigation of the mechanism of duplex-by-duplex recombination catalyzed by vaccinia DNA polymerase. When homologous, blunt-ended linear duplex substrates are incubated with vaccinia polymerase, in the presence of Mg²⁺ and dNTPs, the appearance of joint molecules is preceded by the exposure of complementary single-stranded sequences by the proofreading exonuclease. These intermediates anneal to form a population of joint molecules containing hybrid regions flanked by nicks, 1–5 nt gaps, and/or short overhangs. The products are relatively resistant to exonuclease (and polymerase) activity and thus accumulate in joining reactions. Surface plasmon resonance (SPR) measurements showed the enzyme has a relative binding affinity favoring blunt-ended duplexes over molecules bearing 3′-recessed gaps. Recombinant duplexes are the least favored ligands. These data suggest that a particular combination of otherwise ordinary enzymatic and DNA-binding properties, enable poxvirus DNA polymerases to promote duplex joining reactions.     

Broad activation of the glomerular layer enhances subsequent olfactory responses

Early olfactory experience with a specific odorant enhances the subsequent response of the glomerular layer of the rat olfactory bulb to that same odorant. Because different odorants activate different glomerular layer regions, it seemed plausible that experience with a large number of odorants might result in enhanced glomerular activation during subsequent exposure to both the previously experienced odorants and the novel odorants evoking activity in regions that overlapped with those previously stimulated by different odorants. To this end, 7 odorants were selected using our glomerular response data archive that together stimulated much of the glomerular layer (alpha-phellandrene, benzaldehyde, L-carvone, decanal, pentanol, santalol, and valeric acid). Young rats were exposed to a different odorant each day for 7 days, and this cycle was repeated 3 times from postnatal days 1-21. The [(14)C]2-deoxyglucose technique was used to measure neural activity in response to both previously experienced and novel odorants. The 2 novel odorants (alpha-ionone and L-menthone) activate regions of the glomerular layer that overlap with those stimulated by the 7 enrichment odorants. Our results indicate that early experience with multiple odorants results in increased responsiveness both to previously experienced odorants and to novel odorants that stimulate previously activated regions of the bulb.     

A HPLC-mass spectrometric method suitable for the therapeutic drug monitoring of everolimus

We report here the validation of an HPLC-electrospray-tandem mass spectrometry method for the quantification of everolimus, an immunosuppressant drug. Whole blood samples (100 microl) were extracted by protein precipitation which involved sample pre-treatment with zinc sulphate followed by acetonitrile (containing internal standard, 40-O-(3'-hydroxy)propyl-rapamycin). HPLC was performed using a step-gradient at a flow rate of 0.6 ml/min on a Waters TDM C18 column (10 mm x 2.1mm I.D.) with a resultant chromatographic analysis time of 2 min. Mass spectrometric detection by selected reaction monitoring (everolimus m/z 975.5-->908.3; internal standard m/z 989.5-->922.3). The assay was linear from 0.5 to 40 microg/l (r2>0.994, n=11). The inter- and intra-day analytical recovery and imprecision for quality control samples (1.25, 12.5 and 30 microg/l) were 93.4-98.2% and <10.7%, respectively (n=10). At the lower limit of quantification (0.5 microg/l) the inter- and intra-day analytical recovery was 94.4-95.8% with imprecision of <14.1% (n=10). The absolute recovery of everolimus (6.5 microg/l) and internal standard (12.5 microg/l) was 96.5 and 88.3%, respectively (n=3). A comparison of our method against the mean of all HPLC methods for a series of samples from an external proficiency testing scheme revealed good correlation as shown by the regression analysis: y=0.973x+0.301 (r2=0.986, n=71). In conclusion, the method described is suited to the current requirements for therapeutic drug monitoring of everolimus.     

Thermal denaturations of staphylococcal nuclease wild-type and mutants monitored by fluorescence and circular dichroism are similar: lack of evidence for other than a two state thermal denaturation

It is unclear whether the thermal denaturation of staphylococcal nuclease is a two state, three state, or variable two state process. The thermal denaturation of wild-type staphylococcal nuclease was followed by tryptophan fluorescence and circular dichroism signal at 222 nm, forty-two and fourteen times, respectively. Analysis of this data using a simple two state model gave melting temperatures of 53.0+/-0.4 degrees C (fluorescence) and 52.7+/-0.6 degrees C (CD) and van't Hoff enthalpies of 82.4+/-2.6 kcal/mol and 88.6+/-4.2 kcal/mol. Ninety-seven mutants also had these parameters determined by both fluorescence and CD. The average difference between the melting temperatures was 1.05+/-0.75 degrees and the average difference between van't Hoff enthalpies was 1.6+/-4.8 kcal/mol. These very similar results for the two spectroscopic probes of structure are discussed in the context of the different models that have been proposed for nuclease denaturation. It is concluded, for most nuclease variants, that the errors introduced by a two state assumption are negligible and either virtually all helical structure is lost in any initial unfolding event or any intermediate must have low stability.     

Picornavirus internal ribosome entry site elements can stimulate translation of upstream genes

Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.     

Candidate loci reveal genetic differentiation between temporally divergent migratory runs of Chinook salmon (Oncorhynchus tshawytscha)

Local adaptation is a dynamic process driven by selection that can vary both in space and time. One important temporal adaptation for migratory animals is the time at which individuals return to breeding sites. Chinook salmon (Oncorhynchus tshawytscha) are excellent subjects for studying the genetic basis of temporal adaptation because their high seasonal homing fidelity promotes reproductive isolation leading to the formation of local populations across diverse environments. We tested for adaptive genetic differentiation between seasonal runs of Chinook salmon using two candidate loci; the circadian rhythm gene, OtsClock1b, and Ots515NWFSC, a microsatellite locus showing sequence identity to three salmonid genes central to reproductive development. We found significant evidence for two genetically distinct migratory runs in the Feather River, California (OtsClock1b: F(ST)=0.042, P=0.02; Ots515NWFSC: F(ST)=0.058, P=0.003). In contrast, the fall and threatened spring runs are genetically homogenous based on neutral microsatellite data (F(ST)=-0.0002). Similarly, two temporally divergent migratory runs of Chinook salmon from New Zealand are genetically differentiated based on polymorphisms in the candidate loci (OtsClock1b: F(ST)=0.083, P-value=0.001; Ots515NWFSC: F(ST)=0.095, P-value=0.000). We used an individual-based assignment method to confirm that these recently diverged populations originated from a single source in California. Tests for selective neutrality indicate that OtsClock1b and Ots515NWFSC exhibit substantial departures from neutral expectations in both systems. The large F(ST )estimates could therefore be the result of directional selection. Evidence presented here suggests that OtsClock1b and Ots515NWFSC may influence migration and spawning timing of Chinook salmon in these river systems.