Behavioral and neurochemical changes in the dopaminergic system after repeated cocaine administration

In order to determine whether repeated cocaine administration produced persistent changes in dopamine (DA) receptor binding and release consistent with behavioral sensitization, rats were treated with either cocaine (25 mg/kg ip) or saline twice daily for 14 consecutive days followed by a 3-d withdrawal period. The DA transporter site was assayed using [³H]GBR 12935, whereas D₁ and D₂ sites were assayed using [³H]SCH 23390 and [³H]spiperone, respectively. The density (B _max) of the DA transporter binding sites in the ST of the cocaine-treated group increased significantly (p<0.05) over controls 3 d after the last injection, whereas the density of striatal D₁ and D₂ binding sites remained unchanged. The DA transporter in the nucleus accumbens (NA) was also studied with [³H]GBR 12935 and was unchanged following drug treatment. D₁ and D₂ binding parameters for the NA were not determined in this study. Furthermore, cocaine administration did not affect the affinities (K _d ) of the radioligands used to label the transporter, D₁, or D₂ sites in any of the studies performed. In addition, striatal DA release was measured using in vivo microdialysis in anesthetized rats. Linear regression analysis on maximal decreases in DA release after apomorphine (0.02, 0.2, and 2.0 mg/kg sc) injection showed no difference in the functional capacity of the ST to modulate DA transmission between control and treated groups. Moreover, animals pretreated with cocaine showed a significant (p<0.01) decrease in locomotor activity (LA) after a presynaptic, autoregulating dose of apomorphine (0.03 mg/kg sc) was given. These results suggest that the effects seen after repeated exposure to cocaine may be regulated, in part, by changes in striatal DA transporter binding site densities and not necessarily by DA-releasing mechanisms or D₁ and D₂ receptor modification.     

Carvedilol and its new analogs suppress arrhythmogenic store overload-induced Ca2+ release

Carvedilol is one of the most effective beta blockers for preventing ventricular tachyarrhythmias in heart failure, but the mechanisms underlying its favorable antiarrhythmic benefits remain unclear. Spontaneous Ca(2+) waves, also called store overload-induced Ca(2+) release (SOICR), evoke ventricular tachyarrhythmias in individuals with heart failure. Here we show that carvedilol is the only beta blocker tested that effectively suppresses SOICR by directly reducing the open duration of the cardiac ryanodine receptor (RyR2). This unique anti-SOICR activity of carvedilol, combined with its beta-blocking activity, probably contributes to its favorable antiarrhythmic effect. To enable optimal titration of carvedilol's actions as a beta blocker and as a suppressor of SOICR separately, we developed a new SOICR-inhibiting, minimally beta-blocking carvedilol analog, VK-II-86. VK-II-86 prevented stress-induced ventricular tachyarrhythmias in RyR2-mutant mice and did so more effectively when combined with either of the selective beta blockers metoprolol or bisoprolol. Combining SOICR inhibition with optimal beta blockade has the potential to provide antiarrhythmic therapy that can be tailored to individual patients.     

Removal of FKBP12.6 does not alter the conductance and activation of the cardiac ryanodine receptor or the susceptibility to stress-induced ventricular arrhythmias

The 12.6-kDa FK506-binding protein (FKBP12.6) is considered to be a key regulator of the cardiac ryanodine receptor (RyR2), but its precise role in RyR2 function is complex and controversial. In the present study we investigated the impact of FKBP12.6 removal on the properties of the RyR2 channel and the propensity for spontaneous Ca(2+) release and the occurrence of ventricular arrhythmias. Single channel recordings in lipid bilayers showed that FK506 treatment of recombinant RyR2 co-expressed with or without FKBP12.6 or native canine RyR2 did not induce long-lived subconductance states. [(3)H]Ryanodine binding studies revealed that coexpression with or without FKBP12.6 or treatment with or without FK506 did not alter the sensitivity of RyR2 to activation by Ca(2+) or caffeine. Furthermore, single cell Ca(2+) imaging analyses demonstrated that HEK293 cells co-expressing RyR2 and FKBP12.6 or expressing RyR2 alone displayed the same propensity for spontaneous Ca(2+) release or store overload-induced Ca(2+) release (SOICR). FK506 increased the amplitude and decreased the frequency of SOICR in HEK293 cells expressing RyR2 with or without FKBP12.6, indicating that the action of FK506 on SOICR is independent of FKBP12.6. As with recombinant RyR2, the conductance and ligand-gating properties of single RyR2 channels from FKBP12.6-null mice were indistinguishable from those of single wild type channels. Moreover, FKBP12.6-null mice did not exhibit enhanced susceptibility to stress-induced ventricular arrhythmias, in contrast to previous reports. Collectively, our results demonstrate that the loss of FKBP12.6 has no significant effect on the conduction and activation of RyR2 or the propensity for spontaneous Ca(2+) release and stress-induced ventricular arrhythmias.     

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells

Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.     

Endonuclease-like activity of heme proteins

Heme proteins, metmyoglobin, methemoglobin, and metcytochrome c showed unusual affinity for double-stranded DNA. Calorimetric studies show that binding of methemoglobin to calf thymus DNA (CTDNA) is weakly endothermic, and the binding constant is 4.9+/-0.7x10(5) M(-1). The Soret absorption bands of the heme proteins remained unchanged, in the presence of excess CTDNA, but a new circular dichroic band appeared at 210 nm. Helix melting studies indicated that the protein-DNA mixture denatures at a lower temperature than the individual components. Thermograms obtained by differential scanning calorimetry of the mixture indicated two distinct transitions, which are comparable to the thermograms obtained for individual components, but there was a reduction in the excess heat capacity. Activation of heme proteins by hydrogen peroxide resulted in the formation of high valent Fe(IV) oxo intermediates, and CTDNA reacted rapidly under these conditions. The rate was first-order in DNA concentration, and this reactivity resulted in DNA strand cleavage. Upon activation with hydrogen peroxide, for example, the heme proteins converted the supercoiled pUC18 DNA into nicked circular and linear DNA. No reaction occurred in the absence of the heme protein, or hydrogen peroxide. These data clearly indicate a novel property of several heme proteins, and this is first report of the endonuclease-like activity of the heme proteins.     

Thermolytic release of covalently linked DNA oligonucleotides and their conjugates from controlled-pore glass at near neutral pH

The functionalization of long chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and its conversion to the support 7 has led to the synthesis of DNA oligonucleotides and their 3'- or (3',5')-conjugates. Indeed, CPG support 7 has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. Unlike conventional succinylated CPG supports, this distinctively functionalized support allows oligonucleotide deprotection and removal of the deprotection side products to proceed without releasing the oligonucleotide into the aqueous milieu. When freed from deprotection side products, the DNA oligonucleotide is thermolytically released from the support within 2 h under nearly neutral conditions (pH 7.2, 90 degrees C). The quality of these oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated CPG supports in terms of shorter than full length oligonucleotide contaminants and overall yields. The versatility of the thermolytic CPG support 7 is further demonstrated by the synthesis of a DNA oligonucleotide (20-mer) and its conjugation with an azido and alkynyl groups at both 5'-and 3'-termini, respectively. The functionality of the (3',5')-heteroconjugated oligonucleotide 18 is verified by its circularization to the DNA oligonucleotide 19 under "click" chemistry conditions.     

Reference map for liquid chromatography–mass spectrometry-based quantitative proteomics

The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography–mass spectrometry (LC–MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC–tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.