Bipolar mania and epilepsy pathophysiology and treatment may converge in purine metabolism: A new perspective on available evidence.

Decreased ATPergic signaling is an increasingly recognized pathophysiology in bipolar mania disease models. In parallel, adenosine deficit is increasingly recognized in epilepsy pathophysiology. Under-recognized ATP and/or adenosine-increasing mechanisms of several antimanic and antiseizure therapies including lithium, valproate, carbamazepine, and ECT suggest a fundamental pathogenic role of adenosine deficit in bipolar mania to match the established role of adenosine deficit in epilepsy. The depletion of adenosine-derivatives within the purine cycle is expected to result in a compensatory increase in oxopurines (uric acid precursors) and secondarily increased uric acid, observed in both bipolar mania and epilepsy. Cortisol-based inhibition of purine conversion to adenosine-derivatives may be reflected in observed uric acid increases and the well-established contribution of cortisol to both bipolar mania and epilepsy pathology. Cortisol-inhibited conversion from IMP to AMP as precursor of both ATP and adenosine may represent a mechanism for treatment resistance common in both bipolar mania and epilepsy. Anti-cortisol therapies may therefore augment other treatments both in bipolar mania and epilepsy. Evidence linking (i) adenosine deficit with a decreased need for sleep, (ii) IMP/cGMP excess with compulsive hypersexuality, and (iii) guanosine excess with grandiose delusions may converge to suggest a novel theory of bipolar mania as a condition characterized by disrupted purine metabolism. The potential for disease-modification and prevention related to adenosine-mediated epigenetic changes in epilepsy may be mirrored in mania. Evaluating the purinergic effects of existing agents and validating purine dysregulation may improve diagnosis and treatment in bipolar mania and epilepsy and provide specific targets for drug development.

Hearing loss alters serotonergic modulation of intrinsic excitability in auditory cortex.

Sensorineural hearing loss during early childhood alters auditory cortical evoked potentials in humans and profoundly changes auditory processing in hearing-impaired animals. Multiple mechanisms underlie the early postnatal establishment of cortical circuits, but one important set of developmental mechanisms relies on the neuromodulator serotonin (5-hydroxytryptamine [5-HT]). On the other hand, early sensory activity may also regulate the establishment of adultlike 5-HT receptor expression and function. We examined the role of 5-HT in auditory cortex by first investigating how 5-HT neurotransmission and 5-HT(2) receptors influence the intrinsic excitability of layer II/III pyramidal neurons in brain slices of primary auditory cortex (A1). A brief application of 5-HT (50 µM) transiently and reversibly decreased firing rates, input resistance, and spike rate adaptation in normal postnatal day 12 (P12) to P21 rats. Compared with sham-operated animals, cochlear ablation increased excitability at P12-P21, but all the effects of 5-HT, except for the decrease in adaptation, were eliminated in both sham-operated and cochlear-ablated rats. At P30-P35, cochlear ablation did not increase intrinsic excitability compared with shams, but it did prevent a pronounced decrease in excitability that appeared 10 min after 5-HT application. We also tested whether the effects on excitability were mediated by 5-HT(2) receptors. In the presence of the 5-HT(2)-receptor antagonist, ketanserin, 5-HT significantly decreased excitability compared with 5-HT or ketanserin alone in both sham-operated and cochlear-ablated P12-P21 rats. However, at P30-P35, ketanserin had no effect in sham-operated and only a modest effect cochlear-ablated animals. The 5-HT(2)-specific agonist 5-methoxy-N,N-dimethyltryptamine also had no effect at P12-P21. These results suggest that 5-HT likely regulates pyramidal cell excitability via multiple receptor subtypes with opposing effects. These data also show that early sensorineural hearing loss affects the ability of 5-HT receptor activation to modulate A1 pyramidal cell excitability.

Urinary amino acid analysis: a comparison of iTRAQ-LC-MS/MS, GC-MS, and amino acid analyzer.

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of propyl chloroformate and iTRAQ derivatized amino acids, respectively, to conventional amino acid analysis. The GC-MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC-MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC-MS, and iTRAQ-LC-MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27+/-5.22, 21.18+/-10.94, and 18.34+/-14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39+/-5.35, 6.23+/-3.84, and 35.37+/-29.42. Both GC-MS and iTRAQ-LC-MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.

Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.

We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.

Quantitative chemical proteomics for identifying candidate drug targets.

We have developed a systematic strategy for drug target identification. This consists of the following sequential steps: (1) enrichment of total binding proteins using two differential affinity matrixes upon which are immobilized positive and negative chemical structures for drug activity, respectively; (2) covalent labeling of the proteins with a new cleavable isotope-coded affinity tag (ICAT) reagent, followed by proteolysis of the combined proteins; (3) isolation, identification, and relative quantification of the tagged peptides by liquid chromatography-mass spectrometry; (4) array-based transcription profiling to select candidate proteins; and (5) confirmation of direct interaction between the activity-associated structure and the selected proteins by using surface plasmon resonance. We present a typical application to identify the primary binding protein of a novel class of anticancer agents exemplified by E7070. Our results suggest that this approach provides a new aspect of quantitative proteomics to find specific binding proteins from protein mixture and should be applicable to a wide variety of biologically active small molecules with unidentified target proteins.