Pimavanserin augments the efficacy of atypical antipsychotic drugs in a mouse model of treatment-refractory negative symptoms of schizophrenia.

Negative symptoms are a core, pervasive, and often treatment-refractory phenotype of schizophrenia, one which contributes to poor functional outcome, ability to work, pursue educational goals, and quality of life, as well as caretaker burden. Improvement of negative symptoms in some patients with schizophrenia has been reported with some atypical antipsychotic drugs [AAPDs], but improvement is absent in many patients and partial in others. Therefore, more effective treatments are needed, and better preclinical models of negative symptoms are needed to identify them. Sub-chronic [sc] treatment of rodents with phencyclidine [PCP], a noncompetitive N-methyl-d-aspartate [NMDAR] antagonist, produces deficits in social interactions [SI] that have been widely studied as a model of negative symptoms in schizophrenia. Acute restraint stress [ARS] also provides a model of treatment-refractory negative symptoms [TRS] to AAPDs. By themselves, in sc-PCP mice, the AAPDs, risperidone, olanzapine, and aripiprazole, but not the selective 5-HT2AR inverse agonist, pimavanserin [PIM], rescued the SI deficit in sc-PCP mice, as did the combination of PIM with sub-effective doses of each of these AAPDs. These three AAPDs alone did not rescue SI deficit in sc-PCP+ 2 h-ARS mice, indicating these mice were treatment refractory. However, co-administration of PIM with any of the AAPDs significantly restored SI in these mice. PIM may be an effective adjunctive therapy for treating negative symptoms of schizophrenia in some patients who have failed to respond to AAPDs, but further studies are needed.

Regulation of NF-κB activity by competition between RelA acetylation and ubiquitination.

The nuclear factor (NF)-κB transcription factor has essential roles in inflammation and oncogenesis. Its ubiquitous RelA subunit is regulated by several post-translational modifications, including phosphorylation, ubiquitination and acetylation. Ubiquitination promotes the termination of RelA-dependent transcription, but its regulation is incompletely understood. Through mass spectrometry analysis of ubiquitinated RelA, we identified seven lysines that were attached to degradative and non-degradative forms of polyubiquitin. Interestingly, lysines targeted for acetylation were among the residues identified as ubiquitin acceptor sites. Mutation of these particular sites resulted in decreased polyubiquitination. Acetylation and ubiquitination were found to inhibit each other, consistent with their use of overlapping sites. Reconstitution of rela(-/-) fibroblasts with wild-type and mutant forms of RelA revealed that modifications at these residues can have activating and inhibitory functions depending on the target gene context. Altogether, this study elucidates that ubiquitination and acetylation can modulate each other and regulate nuclear NF-κB function in a gene-specific manner.

Tightening McDonald cerclage suture under sonographic guidance.

OBJECTIVE: The technical factors contributing to failure of cerclage are not fully understood. The aims of this study were to assess the possibility of tightening the McDonald cerclage under ultrasound guidance and to examine the width and shape of the cervical canal before and after tightening the suture. METHODS: A prospective study was performed. The sole indication for cerclage placement was clinical history of cervical insufficiency. Cervical length and canal width were measured by transvaginal ultrasound, at 12-14 weeks' gestation, with the patient's bladder empty, after which the cerclage was performed. Tightening of the suture was performed under sonographic guidance (transabdominal or transrectal) until the cervical canal disappeared from view. After tying the suture, cervical length and the canal width were assessed sonographically. RESULTS: Fifty-eight patients were enrolled in the study; 50 patients had singleton pregnancies and eight patients carried twins. The mean cervical length at the beginning of the procedure was 31 +/- 13 mm (median 30 mm, range 15-48 mm). The mean cervical canal width was 2.1 +/- 0.9 mm (median 2.0 mm, range 0.9-4.5 mm). The mean addition to the length of the cervical canal after the procedure was 11 +/- 0.8 mm (median 1.0, range 8-19 mm). No complications were noted during the procedures. An interesting sonographic finding was an hourglass shape of the cervical canal after the procedure in 16 patients. Of 58 patients, 47 delivered at term, 10 delivered preterm and one miscarried at 18 weeks. Nine of 10 patients with preterm delivery had an hourglass-shaped sonographic appearance of the cervical canal after the procedure. CONCLUSIONS: McDonald cerclage can be tightened under ultrasound guidance. The sonographic appearance of an hourglass shape of the cervical canal following suture tightening may be a risk factor for preterm delivery.

Investigation of chimerical and tagged forms of recombinant rat nucleoside diphosphate kinases alpha and beta. Interaction with rhodopsin-transducin complex and thermal stability.

To elucidate the physicochemical basis of differences between the isoforms of mammalian multifunctional nucleoside diphosphate kinase (NDP), we investigated the recombinant rat homohexameric NDP kinases alpha and beta, consisting of highly homologous alpha or beta subunits of 152 residues each and differing only in variable regions V1 and V2, and their chimerical forms (NDP kinase alpha(1-130)beta(131-152) and NDP kinase beta(1-130)alpha(131-152)) and tagged derivatives (NDP kinase HA-alpha(1-130)beta(131-152), NDP kinase HA-beta(1-130)alpha(131-152), and NDP kinase HA-beta). The thermal stability of these proteins and the ability of some of them to interact with the rhodopsin-transducin (R*Gt) complex have been studied. It was found that NDP kinase alpha, NDP kinase alpha(1-130)beta(131-152), and NDP kinase HA-alpha(1-130)beta(131-152) were similar in their thermal stability (T(1/2) = 61-63 degrees C). NDP kinase beta, NDP kinase beta(1-130)alpha(131-152), NDP kinase HA-beta(1-130)alpha(131-152), and NDP kinase HA-beta were inactivated at a lower temperature (T(1/2) = 51-54 degrees C). NDP kinase HA-alpha(1-130)beta(131-152) interacted with the R*Gt complex in the same manner as NDP kinase alpha, whereas the interaction of NDP kinase HA-beta(1-130)alpha(131-152) and NDP kinase beta with the photoreceptor membranes under the same conditions was very weak. It is suggested that the variability of the region V1 is a structural basis for the multifunctionality of NDP kinase hexamers in the cell.

Early sonographic prenatal diagnosis of seizures.

Fetal seizures are an unusual phenomenon. When diagnosed by ultrasonography, they are frequently associated with malformations and carry a poor prognosis. We describe first trimester seizures in two siblings with arthrogryposis multiplex congenita. In both cases, convulsions appeared before other sonographic signs of the disease. Review of the literature revealed 11 other cases of fetal seizures diagnosed by ultrasound, all later in gestation. Fetal seizures may be the first manifestation of defective neural and motor development. Therefore, in pregnancies at high risk for neuromuscular disease, early sonographic evaluation of fetal motility, in addition to the anatomical survey, is advised.

COMMD proteins: COMMing to the scene.

COMM Domain-containing or COMMD proteins are a recently discovered group of factors defined by the presence of a unique motif in their extreme carboxy termini (Copper metabolism MURR1, or COMM domain). This protein family is comprised of ten members which are widely conserved throughout evolution and share certain functional properties. At the present time, a number of seemingly discrete functions have been ascribed to these factors. These include the regulation of such events as the activity of the transcription factor NF-kappaB, copper homeostasis, the function of the epithelial sodium channel, and cell proliferation. A unifying mechanism that would explain all these events is lacking at the moment, but recent studies suggest that regulation of the ubiquitin pathway may be the basis of many of the functions of the COMMD protein family.

In vitro pharmacology of clinically used central nervous system-active drugs as inverse H(1) receptor agonists.

The human histamine H(1) receptor (H(1)R) is a prototypical G protein-coupled receptor and an important, well characterized target for the development of antagonists to treat allergic conditions. Many neuropsychiatric drugs are also known to potently antagonize this receptor, underlying aspects of their side effect profiles. We have used the cell-based receptor selection and amplification technology assay to further define the clinical pharmacology of the human H(1)R by evaluating >130 therapeutic and reference drugs for functional receptor activity. Based on this screen, we have reported on the identification of 8R-lisuride as a potent stereospecific partial H(1)R agonist (Mol Pharmacol 65:538-549, 2004). In contrast, herein we report on a large number of varied clinical and chemical classes of drugs that are active in the central nervous system that display potent H(1)R inverse agonist activity. Absolute and rank order of functional potency of these clinically relevant brain-penetrating drugs may possibly be used to predict aspects of their clinical profiles, including propensity for sedation.

Intrinsic efficacy of antipsychotics at human D2, D3, and D4 dopamine receptors: identification of the clozapine metabolite N-desmethylclozapine as a D2/D3 partial agonist.

Drugs that antagonize D2-like receptors are effective antipsychotics, but the debilitating movement disorder side effects associated with these drugs cannot be dissociated from dopamine receptor blockade. The "atypical" antipsychotics have a lower propensity to cause extrapyramidal symptoms (EPS), but the molecular basis for this is not fully understood nor is the impact of inverse agonism upon their clinical properties. Using a cell-based functional assay, we demonstrate that overexpression of Galphao induces constitutive activity in the human D2-like receptors (D2, D3, and D4). A large collection of typical and atypical antipsychotics was profiled for activity at these receptors. Virtually all were D2 and D3 inverse agonists, whereas none was D4 inverse agonist, although many were potent D4 antagonists. The inverse agonist activity of haloperidol at D2 and D3 receptors could be reversed by mesoridazine demonstrating that there were significant differences in the degrees of inverse agonism among the compounds tested. Aripiprazole and the principle active metabolite of clozapine NDMC [8-chloro-11-(1-piperazinyl)-5H-dibenzo [b,e] [1,4] diazepine] were identified as partial agonists at D2 and D3 receptors, although clozapine itself was an inverse agonist at these receptors. NDMC-induced functional responses could be reversed by clozapine. It is proposed that the low incidence of EPS associated with clozapine and aripiprazole used may be due, in part, to these partial agonist properties of NDMC and aripiprazole and that bypassing clozapine blockade through direct administration of NDMC to patients may provide superior antipsychotic efficacy.

The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine.

RATIONALE: Clozapine is a unique antipsychotic, with efficacy against positive symptoms in treatment-resistant schizophrenic patients, and the ability to improve cognition and treat the negative symptoms characteristic of this disease. Despite its unique clinical actions, no specific molecular mechanism responsible for these actions has yet been described. OBJECTIVES AND METHODS: To comprehensively profile a large library of neuropsychiatric drugs, including most antipsychotics, at human monoamine receptors using R-SAT, an in vitro functional assay. RESULTS: Profiling revealed that N-desmethylclozapine (NDMC), the principal metabolite of clozapine, but not clozapine itself, is a potent and efficacious muscarinic receptor agonist, a molecular property not shared by any other antipsychotic. To further explore the role of NDMC muscarinic receptor agonist properties in mediating the physiological actions of clozapine, systemically administered NDMC was found to stimulate the phosphorylation of mitogen-activated protein kinase (MAP kinase) in mouse CA1 hippocampal neurons, an effect that was blocked by scopolamine, confirming central M1 muscarinic receptor agonist activity in vivo. Lastly, an analysis of clozapine and NDMC serum levels in schizophrenic patients indicated that high NDMC/clozapine ratios better predicted improvement in cognitive functioning and quality of life than the levels of either compound alone. CONCLUSIONS: The muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy.

Anemia during pregnancy and treatment with intravenous iron: review of the literature.

Anemia, the decrease of the hemoglobin concentration with a consequent decrease in the hematocrit level, is a common disorder complicating pregnancies and is mostly due to iron deficiency. The increase of iron requirements, plasma volume, and the poor intake of iron constitute the principal causes of this deficiency. The present review summarizes the current literature regarding anemia during pregnancy and the parenteral iron therapy options.

Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor.

The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinct receptor isoforms, of which isoforms 1, 2, and 4, encode functional proteins. Detailed pharmacology on isoforms 1 (unspliced receptor), and 2 (which has an 80 amino acid deletion within the third intracellular loop of the protein) revealed that both isoforms displayed robust responses to a series of known H(3) agonists, while all agonists tested displayed increased potency at isoform 2 relative to isoform 1. Histamine, N(alpha)-methylhistamine, and R(-) and S(+)-alpha-methylhistamine are 16-23-fold more potent, while immepip and imetit are three to fivefold more potent. Antagonist experiments revealed a rank order of potency at both isoforms of clobenpropit>iodophenpropit>thioperamide, and these drugs are fivefold less potent at isoform 2 than isoform 1. To further explore the pharmacology of H(3) receptor function, we screened 150 clinically relevant neuropsychiatric drugs for H(3) receptor activity, and identified a small number of antipsychotics that possess significant antagonist activity.

5-hydroxytryptamine2A receptor inverse agonists as antipsychotics.

We have used a cell-based functional assay to define the pharmacological profiles of a wide range of central nervous system active compounds as agonists, competitive antagonists, and inverse agonists at almost all known monoaminergic G-protein-coupled receptor (GPCR) subtypes. Detailed profiling of 40 antipsychotics confirmed that as expected, most of these agents are potent competitive antagonists of the dopamine D2 receptor. Surprisingly, this analysis also revealed that most are potent and fully efficacious 5-hydroxytryptamine (5-HT)2A receptor inverse agonists. No other molecular property was shared as universally by this class of compounds. Furthermore, comparisons of receptor potencies revealed that antipsychotics with the highest extrapyramidal side effects (EPS) liability are significantly more potent at D2 receptors, the EPS-sparing atypical agents had relatively higher potencies at 5-HT2A receptors, while three were significantly more potent at 5-HT2A receptors. Functional high-throughput screening of a diverse chemical library identified 530 ligands with inverse agonist activity at 5-HT2A receptors, including several series of compounds related to known antipsychotics, as well as a number of novel chemistries. An analog of one of the novel chemical series, AC-90179, was pharmacologically profiled against the remaining monoaminergic GPCRs and found to be a highly selective 5-HT2A receptor inverse agonist. The behavioral pharmacology of AC-90179 is characteristic of an atypical antipsychotic agent.

Decomposition of protein tryptophan fluorescence spectra into log-normal components. I. Decomposition algorithms.

Two algorithms of decomposition of composite protein tryptophan fluorescence spectra were developed based on the possibility that the shape of elementary spectral component could be accurately described by a uniparametric log-normal function. The need for several mathematically different algorithms is dictated by the fact that decomposition of spectra into widely overlapping smooth components is a typical incorrect problem. Only the coincidence of components obtained with various algorithms can guarantee correctness and reliability of results. In this paper we propose the following algorithms of decomposition: (1) the SImple fitting procedure using the root-Mean-Square criterion (SIMS) operating with either individual emission spectra or sets of spectra measured with various quencher concentrations; and (2) the pseudo-graphic analytical procedure using a PHase plane in coordinates of normalized emission intensities at various wavelengths (wavenumbers) and REsolving sets of spectra measured with various Quencher concentrations (PHREQ). The actual experimental noise precludes decomposition of protein spectra into more than three components.

Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins.

The physical causes for wide variation of Stokes shift values in emission spectra of tryptophan fluorophores in proteins have been proposed in the model of discrete states (Burstein, E. A., N. S. Vedenkina, and M. N. Ivkova. 1973. Photochem. Photobiol. 18:263-279; Burstein, E. A. 1977a. Intrinsic Protein Luminescence (The Nature and Application). In Advances in Science and Technology (Itogi Nauki i Tekhniki), Biophysics Vol. 7. VINITI, Moscow [In Russian]; Burstein, E. A. 1983. Molecular Biology (Moscow) 17:455-467 [In Russian; English translation]). It was assumed that the existence of the five most probable spectral classes of emitting tryptophan residues and differences among the classes were analyzed in terms of various combinations of specific and universal interactions of excited fluorophores with their environment. The development of stable algorithms of decomposition of tryptophan fluorescence spectra into log-normal components gave us an opportunity to apply two mathematically different algorithms, SImple fitting with Mean-Square criterion (SIMS) and PHase-plot-based REsolving with Quenchers (PHREQ) for the decomposition of a representative set of emission spectra of proteins. Here we present the results of decomposition of tryptophan emission spectra of >100 different proteins, some in various structural states (native and denatured, in complexes with ions or organic ligands, in various pH-induced conformations, etc.). Analysis of the histograms of occurrence of >300 spectral log-normal components with various maximum positions confirmed the statistical discreteness of several states of emitting tryptophan fluorophores in proteins.

Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues.

In our previous paper (Reshetnyak, Ya. K., and E. A. Burstein. 2001. Biophys. J. 81:1710-1734) we confirmed the existence of five statistically discrete classes of emitting tryptophan fluorophores in proteins. The differences in fluorescence properties of tryptophan residues of these five classes reflect differences in interactions of excited states of tryptophan fluorophores with their microenvironment in proteins. Here we present a system of describing physical and structural parameters of microenvironments of tryptophan residues based on analysis of atomic crystal structures of proteins. The application of multidimensional statistical methods of cluster and discriminant analyses for the set of microenvironment parameters of 137 tryptophan residues of 48 proteins with known three-dimensional structures allowed us to 1) demonstrate the discrete nature of ensembles of structural parameters of tryptophan residues in proteins; 2) assign spectral components obtained after decomposition of tryptophan fluorescence spectra to individual tryptophan residues; 3) find a correlation between spectroscopic and physico-structural features of the microenvironment; and 4) reveal differences in structural and physical parameters of the microenvironment of tryptophan residues belonging to various spectral classes.

Constitutively active muscarinic receptors.

Mutations that increase constitutive activity and alter ligand binding have been used to investigate the structure and mechanism of activation of muscarinic receptors. These data are reviewed with reference to the recently published three-dimensional structure of rhodopsin. Residues in TM3 and TM6 where amino acid substitutions increased constitutive activity align with residues within the core of the receptor. A nucleus of these residues is located immediately below the predicted binding site of acetylcholine. The i2 loop where mutations also increase constitutive activity was found to loop away from the i3 loop, which has been found to modulate G-protein coupling specificity.

The identification of tryptophan residues responsible for ATP-induced increase in intrinsic fluorescence of myosin subfragment 1.

ATP binding to myosin subfragment 1 (S1) induces an increase in tryptophan fluorescence. Chymotryptic rabbit skeletal S1 has 5 tryptophan residues (Trp113, 131, 440, 510 and 595), and therefore the identification of tryptophan residues perturbed by ATP is quite complex. To solve this problem we resolved the complex fluorescence spectra into log-normal and decay-associated components, and carried out the structural analysis of the microenvironment of each tryptophan in S1. The decomposition of fluorescence spectra of S1 and S1-ATP complex revealed 3 components with maxima at ca. 318, 331 and 339-342 nm. The comparison of structural parameters of microenvironment of 5 tryptophan residues with the same parameters of single-tryptophan-containing proteins with well identified fluorescence properties applying statistical method of cluster analysis, enabled us to assign Trp595 to 318 nm, Trp440 to 331 nm, and Trp 13, 131 and 510 to 342 nm spectral components. ATP induced an almost equal increase in the intensities of the intermediate (331 nm) and long-wavelength (342 nm) components, and a small decrease in the short component (318 nm). The increase in the intermediate component fluorescence most likely results from an immobilization of some quenching groups (Met437, Met441 and/or Arg444) in the environment of Trp440. The increase in the intensity and a blue shift of the long component might be associated with conformational changes in the vicinity of Trp510. However, these conclusions can not be extended directly to the other types of myosins due to the diversity in the tryptophan content and their microenvironments.

Functional importance of the Ala(116)-Pro(136) region in the calcium-sensing receptor. Constitutive activity and inverse agonism in a family C G-protein-coupled receptor.

The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca(2+) have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found in the Ala(116)-Pro(136) region of CaR, indicating that this part of the receptor is particularly sensitive to mutation-induced activation. This region was subjected to random saturation mutagenesis, and 219 mutant receptor clones were isolated and screened pharmacologically in a high throughput screening assay. Selected mutants were characterized further in an inositol phosphate assay. The vast majority of the mutants tested displayed an increased affinity for Ca(2+). Furthermore, 21 of the mutants showed increased basal activity in the absence of agonist. This constitutive activity was not diminished when the mutations were transferred to a chimeric receptor Ca/1a consisting of the amino-terminal domain of the CaR and the 7 transmembrane and intracellular domains of the metabotropic glutamate receptor mGluR1a. CPCCOEt, a noncompetitive antagonist acting at the 7 transmembrane domain of mGluR1a, suppressed the elevated basal response of the constitutively activated Ca/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala(116)-Pro(136) region is of key importance for the maintenance of the inactive conformation of CaR.

Comparative study of recombinant rat nucleoside diphosphate kinases alpha and beta by intrinsic protein fluorescence.

Nucleoside diphosphate (NDP) kinases of mammals are hexamers of two sorts of randomly associated highly homologous subunits of 152 residues each and, therefore exist in cell as NDP kinase isoforms. The catalytic properties and three-dimensional structures of the isoforms are very similar. The physiological meaning of the existence of the isoforms in cells remained unclear, but studying recombinant rat NDP kinases alpha and beta, each containing only one sort of subunits, we discovered that, in contrast to the isoenzyme beta, NDP kinase alpha is able to interact with the complex between bleached rhodopsin and G-protein transducin in retinal rod membranes at lowered pH values (Orlov et al. FEBS Lett. 389, 186-190, 1996). In order to search for possible molecular basis of such differences between these isoenzymes, a detailed comparative study of their intrinsic fluorescence properties in a large range of solvent conditions was performed in this work. The isoenzymes alpha and beta both contain the same three tryptophan (Trp78, 133, Ind 149) and four tyrosine (Tyr 52, 67, 147, and 151) residues per subunit, but exhibit pronounced differences in their fluorescence properties (both in spectral positions and shape and quantum yield values) and behave differently under pH titration. Whereas NDP kinase alpha undergoes spectral changes in the pH range 5-7 with the mid-point at 6.2, no unequivocal indication of a structural change of NDP kinase beta under pH titration from 9 to 5 was obtained. Since the pH dependencies obtained for fluorescence of isoenzyme alpha resembles the dependence of its binding to the rhodopsin-transducin complex it was suggested that the differences between the NDP kinase isoenzymes alpha and beta in the pH-induced behavior, revealed by the fluorescence spectroscopy, and the differences in their ability to interact with rhodopsin-transducin complex may have the same physical nature, that would be a physico-chemical reason of possible functional dissimilarity of NDP kinase isoforms in cell. An additional analysis of three-dimensional structure of homologous NDP kinases revealed that the source of the differences in fluorescence properties and pH-titration behavior between the isoenzymes alpha and beta may be due to the difference in their global electrostatic charges, rather than to any structural differences between them at neutral pH. The unusually high positive electrostatic potential at he deeply buried active site Tyr52 makes possible that it exists in deprotonated tyrosinate form at neutral and moderately acidic solution. Such a possibility may account for rather unusual fluorescence properties of NDP kinase alpha: (i) rather long-wavelength emission of NDP kinase alpha at ca. 340 nm at pH ca. 8 at extremely low accessibility to external quenchers and, possibly, (ii) an unusually high quantum yield value (ca. 0.42).