Chemistry-based molecular signature underlying the atypia of clozapine.

The central nervous system is functionally organized as a dynamic network of interacting neural circuits that underlies observable behaviors. At higher resolution, these behaviors, or phenotypes, are defined by the activity of a specific set of biomolecules within those circuits. Identification of molecules that govern psychiatric phenotypes is a major challenge. The only organic molecular entities objectively associated with psychiatric phenotypes in humans are drugs that induce psychiatric phenotypes and drugs used for treatment of specific psychiatric conditions. Here, we identified candidate biomolecules contributing to the organic basis for psychosis by deriving an in vivo biomolecule-tissue signature for the atypical pharmacologic action of the antipsychotic drug clozapine. Our novel in silico approach identifies the ensemble of potential drug targets based on the drug's chemical structure and the region-specific gene expression profile of each target in the central nervous system. We subtracted the signature of the action of clozapine from that of a typical antipsychotic, chlorpromazine. Our results implicate dopamine D4 receptors in the pineal gland and muscarinic acetylcholine M1 (CHRM1) and M3 (CHRM3) receptors in the prefrontal cortex (PFC) as significant and unique to clozapine, whereas serotonin receptors 5-HT2A in the PFC and 5-HT2C in the caudate nucleus were common significant sites of action for both drugs. Our results suggest that D4 and CHRM1 receptor activity in specific tissues may represent underappreciated drug targets to advance the pharmacologic treatment of schizophrenia. These findings may enhance our understanding of the organic basis of psychiatric disorders and help developing effective therapies.

Phenytoin is an estrogen receptor α-selective modulator that interacts with helix 12.

RATIONALE: Phenytoin (Dilantin(®); DPH) is used to treat epilepsy but causes estrogen agonist-antagonist-like side effects. We investigated the interaction of phenytoin with estrogen receptors (ERs) α and ß by computational molecular docking, ER competition binding, transcriptional assays, and biological actions, comparing outcomes with estradiol (E2), estrone (E1), and tamoxifen (TMX). EXPERIMENTAL: (1) The DPH docking to 3-dimensional crystal structures of the ERα ligand-binding domain (LBD) showed a high degree of structural complementarity (-57.15 calculated energy units, approximating kcal/mol) with the ligand-binding pocket, including a contact at leucine (L540) in helix 12. Estrogen receptor ß showed slightly less favorable interactions (-54.27 kcal/mol), without contacting L450. Estradiol, E1, and TMX contact points with ERα and ERß do not include L450. (2) Cellular actions: Incubation of cells transfected with ERα or ERß and a luciferase promoter phenytoin was several orders weaker than E2 as an agonist through ERα and had no effect through ERß. However, phenytoin at clinical concentrations (10(-11) to 10(-6) mol/L) powerfully antagonized action of E2 on ERα-expressing cells. Similarly, phenytoin at clinically effective concentrations marginally induced alkaline phosphatase by ERα- and ERß-expressing endometrial cancer cells but at doses well below clinical effectiveness blocked E2-induced alkaline phosphatase. (3) ER competition: In Scatchard plots comparing phenytoin with 17ß-estradiol against endometrial cancer cell cytosol E2-alone more effectively displaced labeled E2 than phenytoin, but phenytoin was approximately equimolar effective to E2 in inhibiting E2's displacement of the radiolabel, further confirming that phenytoin is a strong E2 antagonist. CONCLUSIONS: At clinically effective concentrations, phenytoin is a strong ERα cell antagonist but a many-fold weaker agonist. Although it interacts with ERß LBD residues, phenytoin has no effects on ERß-only expressing cells. Docking studies indicate phenytoin interacts with the ERα LBD at the hinge of helix 12 and could thereby interfere with the entry of other ER ligands or with the mobility of helix 12, either of which actions could explain phenytoin's antagonism of ER-mediated E2 actions. Our results suggest an explanation for the broad profile of phenytoin's actions and raise possibilities for the use of phenytoin or congeners in the clinical management of ERα-dependent conditions.

Genetic linkage mapping of zebrafish genes and ESTs.

Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes essential for vertebrate development, physiology, and behavior. We have constructed a genetic linkage map that will facilitate the identification of candidate genes for these mutations and allow comparisons among the genomes of zebrafish and other vertebrates. On this map, we have localized 771 zebrafish genes and expressed sequence tags (ESTs) by scoring single-stranded conformational polymorphisms (SSCPs) in a meiotic mapping panel. Of these sequences, 642 represent previously unmapped genes and ESTs. The mapping panel was comprised of 42 homozygous diploid individuals produced by heat shock treatment of haploid embryos at the one-cell stage (HS diploids). This "doubled haploid" strategy combines the advantages of mapping in haploid and standard diploid systems, because heat shock diploid individuals have only one allele at each locus and can survive to adulthood, enabling a relatively large quantity of genomic DNA to be prepared from each individual in the mapping panel. To integrate this map with others, we also scored 593 previously mapped simple-sequence length polymorphisms (SSLPs) in the mapping panel. This map will accelerate the molecular analysis of zebrafish mutations and facilitate comparative analysis of vertebrate genomes.

Estimating local backbone structural deviation in homology models.

After the atomic coordinates themselves, the most important data in a homology model are the spatial reliability estimates associated with each of the atoms (atom annotation). Recent blind homology modeling predictions have demonstrated that principally correct sequence-structure alignments are achievable to sequence identities as low as 25% [Martin, A.C., MacArthur, M.W., Thornton, J.M., 1997. Assessment of comparative modeling in CASP2. Proteins Suppl(1), 14-28]. The locations and extent of spatial deviations in the backbone between correctly aligned homologous protein structures remained very poorly estimated however, and these errors were the cause of errant loop predictions [Abagyan, R., Batalov, S., Cardozo, T., Totrov, M., Webber, J., Zhou, Y., 1997. Homology modeling with internal coordinate mechanics: deformation zone mapping and improvements of models via conformational search. Proteins Suppl(1), 29-37]. In order to derive accurate measures for local backbone deviations, we made a systematic study of static local backbone deviations between homologous pairs of protein structures. We found that 'through space' proximity to gaps and chain termini, local three-dimensional 'density', three-dimensional environment conservation, and B-factor of the template contribute to local deviations in the backbone in addition to local sequence identity. Based on these finding, we have identified the meaningful ranges of values within which each of these parameters correlates with static local backbone deviation and produced a combined scoring function to greatly improve the estimation of local backbone deviations. The optimized function has more than twice the accuracy of local sequence identity or B-factor alone and was validated in a recent blind structure prediction experiment. This method may be used to evaluate the utility of a preliminary homology model for a particular biological investigation (e.g. drug design) or to provide an improved starting point for molecular mechanics loop prediction methods.

The selC-associated SHI-2 pathogenicity island of Shigella flexneri.

Pathogenicity islands are chromosomal gene clusters, often located adjacent to tRNA genes, that encode virulence factors present in pathogenic organisms but absent or sporadically found in related non-pathogenic species. The selC tRNA locus is the site of integration of different pathogenicity islands in uropathogenic Escherichia coli, enterohaemorrhagic E. coli and Salmonella enterica. We show here that the selC locus of Shigella flexneri, the aetiological agent of bacterial dysentery, also contains a pathogenicity island. This pathogenicity island, designated SHI-2 (Shigella island 2), occupies 23.8 kb downstream of selC and contains genes encoding the aerobactin iron acquisition siderophore system, colicin V immunity and several novel proteins. Remnants of multiple mobile genetic elements are present in SHI-2. SHI-2-hybridizing sequences were detected in all S. flexneri strains tested and parts of the island were also found in other Shigella species. SHI-2 may allow Shigella survival in stressful environments, such as those encountered during infection.

A genetic linkage map for zebrafish: comparative analysis and localization of genes and expressed sequences.

Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes with essential functions. To facilitate the identification of candidate genes for these mutations, we have genetically mapped 104 genes and expressed sequence tags by scoring single-strand conformational polymorphisms in a panel of haploid siblings. To integrate this map with existing genetic maps, we also scored 275 previously mapped genes, microsatellites, and sequence-tagged sites in the same haploid panel. Systematic phylogenetic analysis defined likely mammalian orthologs of mapped zebrafish genes, and comparison of map positions in zebrafish and mammals identified significant conservation of synteny. This comparative analysis also identified pairs of zebrafish genes that appear to be orthologous to single mammalian genes, suggesting that these genes arose in a genome duplication that occurred in the teleost lineage after the divergence of fish and mammal ancestors. This comparative map analysis will be useful in predicting the locations of zebrafish genes from mammalian gene maps and in understanding the evolution of the vertebrate genome.

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.

Homology modeling with internal coordinate mechanics: deformation zone mapping and improvements of models via conformational search.

Five models by homology containing insertions and deletions and ranging from 33% to 48% sequence identity to the known homologue, and one high sequence identity (85%) model were built for the CASP2 meeting. For all five low identity targets: (i) our starting models were improved by the Internal Coordinate Mechanics (ICM) energy optimization, (ii) the refined models were consistently better than those built with the automatic SWISS-MODEL program, and (iii) the refined models differed by less than 2% from the best model submitted, as judged by the residue contact area difference (CAD) measure [Abagyan, R.A., Totrov, M.J. Mol. Biol. 268:678-685, 1997]. The CAD measure is proposed for ranking models built by homology instead of global root-mean-square deviation, which is frequently dominated by insignificant yet large contributions from incorrectly predicted fragments or side chains. We demonstrate that the precise identification of regions of local backbone deviation is an independent and crucial step in the homology modeling procedure after alignment, since aligned fragments can strongly deviate from the template at various distances from the alignment gap or even in the ungapped parts of the alignment. We show that a local alignment score can be used as an indicator of such local deviation. While four short loops of the meeting targets were predicted by database search, the best loop 1 target T0028, for which the correct database fragment was not found, was predicted by Internal Coordinate Mechanics global energy optimization at 1.2 A accuracy. A classification scheme for errors in homology modeling is proposed.

Long pentraxins: an emerging group of proteins with diverse functions.

The earliest described pentraxins, C reactive protein (CRP) and serum amyloid P component (SAP), are cytokine-inducible acute phase proteins implicated in innate immunity whose concentrations in the blood increase dramatically upon infection or trauma. The highly conserved family of pentraxins was thought to consist solely of approximately 25 kDa proteins. Recently, several distinct larger proteins have been identified in which only the C-terminal halves show characteristic features of the pentraxin family. One of the recently described "long" pentraxins (TSG-14/PTX3) is inducible by TNF or IL-1 and is produced during the acute phase response. Other newly identified long pentraxins are constitutively expressed proteins associated with sperm-egg fusion (apexin/p50), may function at the neuronal synapse (neuronal pentraxin I, NPI), or may serve yet other, unknown functions (NPII and XL-PXN1). Evidence obtained by molecular modeling and by direct physicochemical analysis suggests that TSG-14 protein retains some characteristic structural features of the pentraxins, including the formation of pentameric complexes.

Motility of vinculin-deficient F9 embryonic carcinoma cells analyzed by video, laser confocal, and reflection interference contrast microscopy.

We have studied the motility of wild-type F9 and vinculin-deficient (5.51) mouse embryonal carcinoma cells. F9 cells extended filopodia at a rate of 61 ( +/- 18) nm/s over a distance of 3.18 (+/- 0.29) microns. In contrast, 5.51 cells exhibited filopodia which extended at a similar speed of 57 (+/- 17) nm/s but over a longer distance of 5.10 (+/- 2.14) microns. Cell-substratum contact areas of both cell types were examined by reflection interference contrast microscopy. Wild-type F9 cells had distinct close contacts (dark gray areas) at the cell periphery, whereas 5.51 cells had only a few light gray pinpoint contacts with the substrate. Confocal microscopy showed alpha-actinin to be localized along actin stress fibers in wild-type cells, and in 5.51 cells stress fibers were absent and alpha-actinin was associated with F-actin in the filopodia. beta 1-integrin, talin, and paxillin were concentrated in focal contacts in wild-type cells, but in 5.51 cells beta 1-integrin and talin were in patches under the plasma membrane and paxillin was diffusely distributed in the cytoplasm. We conclude that changes in cell shape and motility of 5.51 compared to wild-type F9 cells are due to the absence of vinculin even though there may be functions of other focal adhesion complex proteins, e.g., talin, linking the actin cytoskeleton to the plasma membrane.

Homology modeling by the ICM method.

Five models have been built by the ICM method for the Comparative Modeling section of the Meeting on the Critical Assessment of Techniques for Protein Structure Prediction. The targets have homologous proteins with known three-dimensional structure with sequence identity ranging from 25 to 77%. After alignment of the target sequence with the related three-dimensional structure, the modeling procedure consists of two subproblems: side-chain prediction and loop prediction. The ICM method approaches these problems with the following steps: (1) a starting model is created based on the homologous structure with the conserved portion fixed and the nonconserved portion having standard covalent geometry and free torsion angles; (2) the Biased Probability Monte Carlo (BPMC) procedure is applied to search the subspaces of either all the nonconservative side-chain torsion angles or torsion angles in a loop backbone and surrounding side chains. A special algorithm was designed to generate low-energy loop deformations. The BPMC procedure globally optimizes the energy function consisting of ECEPP/3 and solvation energy terms. Comparison of the predictions with the NMR or crystallographic solutions reveals a high proportion of correctly predicted side chains. The loops were not correctly predicted because imprinted distortions of the backbone increased the energy of the near-native conformation and thus made the solution unrecognizable. Interestingly, the energy terms were found to be reliable and the sampling of conformational space sufficient. The implications of this finding for the strategies of future comparative modeling are discussed.

Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell.

A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell-substrate adhesion. We have previously shown that neither uvomorulin (E-cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F-actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins.

Expression and localization of villin, fimbrin, and myosin I in differentiating mouse F9 teratocarcinoma cells.

F9 embryonic carcinoma cells are a multipotent cell line which can be induced to differentiate into cells resembling the visceral endoderm, an extraembryonic absorptive epithelium characterized by apical microvilli. We have examined the role of villin, fimbrin, and myosin I, the major actin-binding proteins in the intestinal and visceral yolk sac microvilli, in the development of epithelial polarity and the assembly of the microvillus cytoskeleton in differentiating F9 cells. By immunoblot analysis villin was first detected at 4 days of differentiation. Confocal microscopy localized villin at Day 4 to the apical surface and by Day 6 to the basolateral surfaces as well. In comparison, fimbrin and myosin I were both present in undifferentiated F9 cells and became associated with the apical surface after villin during differentiation to visceral endoderm. The accumulation of villin, fimbrin, and myosin I at the apical surface in differentiating F9 cells correlated with the appearance of microvilli containing organized actin filament bundles. Two mouse villin cDNAs were isolated and characterized to examine villin expression during F9 differentiation. Mouse villin was encoded by two transcripts (3.8 and 3.4 kb) which differ in their 3'-noncoding region. Both villin mRNAs were first detected by Day 4 of differentiation and their appearance coincided with expression of the visceral endoderm marker alpha-fetoprotein. The pattern of expression and order of accumulation of villin, fimbrin, and myosin I in differentiating F9 cells are common to developing gut and yolk sac epithelium. This suggests that microvillus assembly is directed by a sequence of temporally and spatially regulated localizations of these actin-binding proteins.

La ligadura escalonada de las hemorroides. Nuestra experiencia. Comunicacion preliminar. / Staggered ligation of hemorrhoids. Our experience. Preliminary communication

Los autores describen una nueva tecnica para el tratamiento de las hemorroides, consistente en la ligadura escalonada. Exponen los resultados obtenidos en 24 casos tratados con esta tecnica. La ligadura escalonada posee ventajas sobre los metodos de hemorroidectomia, por lo que la hacen el procedimiento de eleccion