Considering a Potential Role of Linalool as a Mood Stabilizer for Bipolar Disorder.

Epidemiologic studies suggest that the lifetime prevalence of bipolar spectrum disorders ranges from 2.8 to 6.5 percent of the population. To decrease morbidity and mortality associated with disease progression, pharmacologic intervention is indicated for the majority of these patients. While a number of effective treatment regimens exist, many conventional medications have significant side effect profiles that adversely impact patients' short and long-term well-being. It is thus important to continue advancing and improving therapeutic options available to patients. This paper reviews the limitations of current treatments and examines the chemical compound Linalool, an alcohol found in many plant species, that may serve as an effective mood stabilizer. While relatively little is known about Linalool and bipolar disorder, the compound has been shown to have antiepileptic, anti-inflammatory, anxiolytic, anti-depressive, and neurotrophic effects, with mechanisms that are comparable to current bipolar disorder treatment options.

Imatinib and Dasatinib Inhibit Hemangiosarcoma and Implicate PDGFR-ß and Src in Tumor Growth.

Hemangiosarcoma, a natural model of human angiosarcoma, is an aggressive vascular tumor diagnosed commonly in dogs. The documented expression of several receptor tyrosine kinases (RTKs) by these tumors makes them attractive targets for therapeutic intervention using tyrosine kinase inhibitors (TKIs). However, we possess limited knowledge of the effects of TKIs on hemangiosarcoma as well as other soft tissue sarcomas. We report here on the use of the TKIs imatinib and dasatinib in canine hemangiosarcoma and their effects on platelet-derived growth factor receptor ß (PDGFR-ß) and Src inhibition. Both TKIs reduced cell viability, but dasatinib was markedly more potent in this regard, mediating cytotoxic effects orders of magnitude greater than imatinib. Dasatinib also inhibited the phosphorylation of the shared PDGFR-ß target at a concentration approximately 1000 times less than that needed by imatinib and effectively blocked Src phosphorylation. Both inhibitors augmented the response to doxorubicin, suggesting that clinical responses likely will be improved using both drugs in combination; however, dasatinib was significantly (P < .05) more effective in this context. Despite the higher concentrations needed in cell-based assays, imatinib significantly inhibited tumor growth (P < .05) in a tumor xenograft model, highlighting that disruption of PDGFR-ß/PDGF signaling may be important in targeting the angiogenic nature of these tumors. Treatment of a dog with spontaneously occurring hemangiosarcoma established that clinically achievable doses of dasatinib may be realized in dogs and provides a means to investigate the effect of TKIs on soft tissue sarcomas in a large animal model.

The effects of taurolidine alone and in combination with doxorubicin or carboplatin in canine osteosarcoma in vitro.

BACKGROUND: Osteosarcoma (OS) affects over 8000 dogs/year in the United States. The disease usually arises in the appendicular skeleton and metastasizes to the lung. Dogs with localized appendicular disease benefit from limb amputation and chemotherapy but most die within 6-12 months despite these treatments. Taurolidine, a derivative of taurine, has anti-tumor and anti-angiogenic effects against a variety of cancers. The following in vitro studies tested taurolidine as a candidate for adjuvant therapy for canine OS. Tests for p53 protein status and caspase activity were used to elucidate mechanisms of taurolidine-induced cell death. RESULTS: Taurolidine was cytotoxic to osteosarcoma cells and increased the toxicity of doxorubicin and carboplatin in vitro. Apoptosis was greatly induced in cells exposed to 125 µM taurolidine and less so in cells exposed to 250 µM taurolidine. Taurolidine cytotoxicity appeared caspase-dependent in one cell line; with apparent mutant p53 protein. This cell line was the most sensitive to single agent taurolidine treatment and had a taurolidine-dependent reduction in accumulated p53 protein suggesting taurolidine's effects may depend on the functional status of p53 in canine OS. CONCLUSION: Taurolidine's cytotoxic effect appears dependent on cell specific factors which may be explained, in part, by the functional status of p53. Taurolidine initiates apoptosis in canine OS cells and this occurs to a greater extent at lower concentrations. Mechanisms of cell death induced by higher concentrations were not elucidated here. Taurolidine combined with doxorubicin or carboplatin can increase the toxicity of these chemotherapy drugs and warrants further investigation in dogs with osteosarcoma.

Use of a murine cell line for identification of human nitric oxide synthase inhibitors.

INTRODUCTION: Nitric oxide (NO) has been implicated in a wide range of physiological and pathological processes. Low concentrations of this mediator play homeostatic roles, whereas many acute and chronic responses are associated with excessive production of NO. This upregulation is due in part to the induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines in several different cell types, including macrophages and their CNS derivative, microglia. METHODS: The crystal structures of the oxygenase domains of mouse and human iNOS were superimposed using the "align by homology" feature in Sybyl (SYBYL 7.0, Tripos Inc.). NOS isoform expression was assessed by TaqMan, Western blotting, and activity assays. RESULTS: We demonstrate that there is a high degree of three-dimensional overlap between the mouse and human iNOS active centers and propose that the murine isoform can serve as a suitable substitute for the human in assays. We also demonstrate that LPS stimulation of the mouse macrophage cell line RAW 264.7 induces the expression of iNOS, but not nNOS or eNOS, at the levels of mRNA transcription and protein expression. Furthermore, the pharmacology and calcium dependency of the NO formation support the finding that it is due to iNOS alone. Also reported is the demonstration of LPS-induced RAW 264.7 macrophages in simple cell-based and cell-free screening assays for iNOS inhibitors. Both assays were reproducible, as demonstrated by Z' factors of 0.69 and 0.71, and had high signal to noise ratios of 11- and 6-fold for the cell-based and cell-free assay, respectively. DISCUSSION: Our computational analyses indicate that there is a high degree of three-dimensional overlap between the oxygenase domains of human and murine iNOS. This observation together with the selective induction of murine iNOS in RAW 264.7 macrophages demonstrates the potential utility of the mouse iNOS assay to identify inhibitors of the human enzyme.

RGSZ1 interacts with protein kinase C interacting protein PKCI-1 and modulates mu opioid receptor signaling.

Protein kinase C interacting protein (PKCI-1) was identified among the potential interactors from a yeast two hybrid screen of human brain library using N terminal of RGSZ1 as a bait. The cysteine string region, unique to the RZ subfamily, contributes to the observed interaction because PKCI-1 interacted with N-terminus of RGS17 and GAIP, but not with that of RGS2 or RGS7 where cysteine string motif is absent. The interaction between RGSZ1 and PKCI-1 was confirmed by coimmunoprecipitation and immunofluorescence. PKCI-1 and RGSZ1 could be detected by coimmunoprecipitation using 14-3-3 antibody in cells transfected with PKCI-1 or RGSZ1 respectively, but when transfected with PKCI-1 and RGSZ1 together, only RGSZ1 could be detected. Phosphorylation of Galphaz by protein kinase C (PKC) reduces the ability of the RGS to effectively function as GTPase accelerating protein for Galphaz, and interferes with ability of Galphaz to interact with betagamma complex. We investigated the roles of 14-3-3 and PKCI-1 in phosphorylation of Galphaz. Phosphorylation of Galphaz by PKC was inhibited by 14-3-3 and the presence of PKCI-1 did not provide any further inhibition. PKCI-1 interacts with mu opioid receptor and suppresses receptor desensitization and PKC related mu opioid receptor phosphorylation [W. Guang, H. Wang, T. Su, I.B. Weinstein, J.B. Wang, Mol. Pharmacol. 66 (2004) 1285.]. Previous studies have also shown that mu opioid receptor co-precipitates with RGSZ1 and influence mu receptor signaling by acting as effector antagonists [J. Garzon, M. Rodriguez-Munoz, P. Sanchez-Blazquez, Neuropharmacology 48 (2005) 853., J. Garzon, M. Rodriguez-Munoz, A. Lopez-Fando, P. Sanchez-Blazquez Neuropsychopharmacology 30 (2005) 1632.]. Inhibition of cAMP by mu opioid receptor was significantly reduced by RGSZ1 and this effect was enhanced in combination with PKCI-1. Our studies thus provide a link between the previous observations mentioned above and indicate that the major function of PKCI-1 is to modulate mu opioid receptor signaling pathway along with RGSZ1, rather than directly mediating the Galphaz RGSZ1 interaction.

High-throughput screening for the discovery of inhibitors of fatty acid amide hydrolase using a microsome-based fluorescent assay.

Fatty acid amide hydrolase (FAAH) is a membrane-associated enzyme that catalyzes the hydrolysis of several endogenous bioactive lipids, including anandamide (AEA), N-palmitoylethanolamine (PEA), oleamide, and N-oleoylethanolamine (OEA). These fatty acid amides participate in many physiological activities such as analgesia, anxiety, sleep modulation, anti inflammatory responses, and appetite suppression. Because FAAH plays an essential role in controlling the tone and activity of these endogenous bioactive lipids, this enzyme has been implicated to be a drug target for the therapeutic management of pain, anxiety, and other disorders. In an effort to discover FAAH inhibitors, the authors have previously reported the development of a novel fluorescent assay using purified FAAH microsomes as an enzyme source and a fluorogenic substrate, arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA). Herein, the authors have adapted this assay to a high-throughput format and have screened a large library of small organic compounds, identifying a number of novel FAAH inhibitors. These data further verify that this fluorescent assay is sufficiently robust, efficient, and low-cost for the identification of FAAH inhibitory molecules and open this class of enzymes for therapeutic exploration.

A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.

A set of ligation-independent expression vectors system has been developed for co-expression of proteins in Escherichia coli. These vectors contain a strong T7 promoter, different drug resistant genes, and an origin of DNA replication from a different incompatibility group, allowing combinations of these plasmids to be stably maintained together. In addition, these plasmids also contain the lacI gene, a transcriptional terminator, and a 3' polyhistidine (6x His) affinity tag (H6) for easy purification of target proteins. All of these vectors contain an identical transportable cassette flanked by suitable restriction enzyme cleavage sites for easy cloning and shuttling among different vectors. This cassette incorporates a ligation-independent cloning (LIC) site for LIC manipulations, an optimal ribosome binding site for efficient protein translation, and a 6x His affinity tag for protein purification Therefore, any E. coli expression vector of choice can be easily converted to LIC type expression vectors by shuttling the cassette using the restriction enzyme cleavage sites at the ends. We have demonstrated the expression capabilities of these vectors by co-expressing three bacterial (dsbA, dsbG, and Trx) and also two other mammalian proteins (KChIP1 and Kv4.3). We further show that co-expressed KChIP1/Kv4.3 forms soluble protein complexes that can be purified for further studies.

Identification and characterization of small molecule modulators of KChIP/Kv4 function.

Potassium channels and their associated subunits are important contributors to electrical excitability in many cell types. In this study, a yeast two-hybrid assay was used to identify inhibitors such as a diaryl-urea compound (CL-888) that binds to and modulates the formation of the Kv4/KChIP complex. CL-888 altered the apparent affinity of KChIP1 to Kv4.3-N in a Biacore assay, but did not dissociate the two proteins in size-exclusion chromatography experiments. Kv4.2/KChIP1 current amplitude and kinetics were altered with compound exposure, supporting the hypothesis of a compound-induced conformational change in the protein complex. Fluorescence spectroscopy of a unique tryptophan residue in KChIP1 was consistent with compound binding to the protein. Molecular modeling using the KChIP1 crystal structure indicates that compound binding may occur in a small tryptophan-containing binding pocket located on the hydrophilic side of the protein.

Two N-terminal domains of Kv4 K(+) channels regulate binding to and modulation by KChIP1.

The family of calcium binding proteins called KChIPs associates with Kv4 family K(+) channels and modulates their biophysical properties. Here, using mutagenesis and X-ray crystallography, we explore the interaction between Kv4 subunits and KChIP1. Two regions in the Kv4.2 N terminus, residues 7-11 and 71-90, are necessary for KChIP1 modulation and interaction with Kv4.2. When inserted into the Kv1.2 N terminus, residues 71-90 of Kv4.2 are also sufficient to confer association with KChIP1. To provide a structural framework for these data, we solved the crystal structures of Kv4.3N and KChIP1 individually. Taken together with the mutagenesis data, the individual structures suggest that that the Kv4 N terminus is required for stable association with KChIP1, perhaps through a hydrophobic surface interaction, and that residues 71-90 in Kv4 subunits form a contact loop that mediates the specific association of KChIPs with Kv4 subunits.

Snapin interacts with the N-terminus of regulator of G protein signaling 7.

The N-terminus of regulator of G protein signaling 7 (RGS7) contains a dishevelled/egl-10/pleckstrin (DEP) domain of unknown function. To gain insight into its function, we used yeast two-hybrid analysis to screen a human whole brain cDNA library in order to identify proteins that interact specifically with the N-terminus of human RGS7 (amino acid residues 1-248). From this analysis, we identified snapin, a protein associated with the SNARE complex in neurons, as an interactor with the N-terminus of RGS7. Deletion mutation analysis in yeast demonstrated that the interaction between RGS7 and snapin is specific and is mediated primarily by amino acid residues 1-69 of RGS7 (which contains the proximal portion of the DEP domain). The interaction between RGS7 and snapin was also demonstrated in mammalian cells by coimmunoprecipitation and pull-down assays. Our results suggest that RGS7 could play a role in synaptic vesicle exocytosis through its interaction with snapin.

A unified model for apical caspase activation.

Apoptosis is orchestrated by the concerted action of caspases, activated in a minimal two-step proteolytic cascade. Existing data suggests that apical caspases are activated by adaptor-mediated clustering of inactive zymogens. However, the mechanism by which apical caspases achieve catalytic competence in their recruitment/activation complexes remains unresolved. We explain that proximity-induced activation of apical caspases is attributable to dimerization. Internal proteolysis does not activate these apical caspases but is a secondary event resulting in partial stabilization of activated dimers. Activation of caspases-8 and -9 occurs by dimerization that is fully recapitulated in vitro by kosmotropes, salts with the ability to stabilize the structure of proteins. Further, single amino acid substitutions at the dimer interface abrogate the activity of caspases-8 and -9 introduced into recipient mammalian cells. We propose a unified caspase activation hypothesis whereby apical caspases are activated by dimerization of monomeric zymogens.

Impact of mobility on structure-based drug design for the MMPs.

Structure-based approaches for drug design generally do not incorporate solvent effects and dynamic information to predict inhibitor-binding affinity because of practical limitations. The matrix metalloproteinases (MMPs) have previously been demonstrated to exhibit significant mobility in their active sites. This dynamic characteristic significantly complicates the drug design process based on static structures, which was clearly observed for a class of hydroxamic acids containing a butynyl moiety. Compound 1 was expected to be selective against MMP-1 based on predicted steric clashes between the butynyl P1' group and the S1' pocket, but the observation of complex inhibitor dynamics in the NMR structure of MMP-1:1 provides an explanation for the low nanomolar binding to MMP-1.

Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling.

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein alpha subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Galpha(z) subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Galpha(z,) but also with Galpha(i), as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind (35)S-labeled Galpha(i1) protein in an AlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an alpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Galpha(i) and regulates Galpha(i)-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.