Therapeutic potential of diosmin, a citrus flavonoid against arsenic-induced neurotoxicity via suppression of NOX 4 and its subunits.

OBJECTIVES: Water contaminated with arsenic affected millions of people worldwide and arsenic exposure is related to various neurological disorders. Hence, the current study was planned to investigate the neuroprotective activity of diosmin (DSN) against arsenic induced neurotoxicity as an attempt to identify therapeutic intervention to combat arsenicism. MATERIALS AND METHODS: Sodium arsenite an inducer of neurotoxicity was administered orally (13 mg/kg) and DSN treatment at two selected doses (50 and 100 mg/kg) was done for 21 days. Behavioral and biochemical variations were examined by various parameters. Furthermore, histopathological and immunohistochemistry studies were done with the brain sections. RESULTS: The behavioral studies evidenced that arsenic has suppressed the exploratory behavior and motor coordination in rats and DSN treatment has recovered the behavioral changes to normal. Arsenic administration has also found to induce oxidative stress and DSN co-treatment has ameliorated the oxidative stress markers. Interestingly, depleted levels of neurotransmitters were observed with the arsenic and it was restored back by the DSN treatment. Histopathological alterations like pyknosis of the neuronal cells were identified with arsenic exposure and subsided upon DSN co administration. Immunohistochemical studies have revealed the expression of NOX4 and its gp91phox and P47phox subunits and its suppression by DSN treatment may be the key therapeutic factor of it. CONCLUSIONS: Treatment with DSN showed a beneficial effect in protecting against arsenic-induced neurotoxicity by suppressing the toxicity changes and the antioxidant effect of DSN might be attributed to its ability of suppressing NOX4 and its subunits.

Discovery of selective fragment-sized immunoproteasome inhibitors.

Proteasomes contribute to maintaining protein homeostasis and their inhibition is beneficial in certain types of cancer and in autoimmune diseases. However, the inhibition of the proteasomes in healthy cells leads to unwanted side-effects and significant effort has been made to identify inhibitors specific for the immunoproteasome, especially to treat diseases which manifest increased levels and activity of this proteasome isoform. Here, we report our efforts to discover fragment-sized inhibitors of the human immunoproteasome. The screening of an in-house library of structurally diverse fragments resulted in the identification of benzo[d]oxazole-2(3H)-thiones, benzo[d]thiazole-2(3H)-thiones, benzo[d]imidazole-2(3H)-thiones, and 1-methylbenzo[d]imidazole-2(3H)-thiones (with a general term benzoXazole-2(3H)-thiones) as inhibitors of the chymotrypsin-like (ß5i) subunit of the immunoproteasome. A subsequent structure-activity relationship study provided us with an insight regarding growing vectors. Binding to the ß5i subunit was shown and selectivity against the ß5 subunit of the constitutive proteasome was determined. Thorough characterization of these compounds suggested that they inhibit the immunoproteasome by forming a disulfide bond with the Cys48 available specifically in the ß5i active site. To obtain fragments with biologically more tractable covalent interactions, we performed a warhead scan, which yielded benzoXazole-2-carbonitriles as promising starting points for the development of selective immunoproteasome inhibitors with non-peptidic scaffolds.

Synthesis and biological evaluation of solubilized sulfonamide analogues of the phosphatidylinositol 3-kinase inhibitor ZSTK474.

Replacing one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 with a variety of sulfonamide-linked solubilizing substituents produced a new class of active and potent PI3Kα inhibitors, with several derivatives demonstrating high PI3Kα enzyme potency and good cellular potency in two human derived cell lines. The overall results suggest a preference for linear and somewhat flexible solubilizing functions. From this series, compound 16, also known as SN32976, was selected for advanced preclinical evaluation.

Identification of potential inhibitors against nuclear Dam1 complex subunit Ask1 of Candida albicans using virtual screening and MD simulations.

Identification of hit compounds against specific target form the starting point for a drug discovery program. A consistent decline of new chemical entities (NCEs) in recent years prompted a challenge to explore newer approaches to discover potential hit compounds that in turn can be converted into leads, and ultimately drug with desired therapeutic efficacy. The vast amount of omics and activity data available in public databases offers an opportunity to identify novel targets and their potential inhibitors. State of the art in silico methods viz., clustering of compounds, virtual screening, molecular docking, MD simulations and MMPBSA calculations were employed in a pipeline to identify potential 'hits' against those targets as well whose structures, as of now, could only predict through threading approaches. In the present work, we have started from scratch, amino acid sequence of target and compounds retrieved from PubChem compound database, modeled it in such a way that led to the identification of possible inhibitors of Dam1 complex subunit Ask1 of Candida albicans. We also propose a ligand based binding site determination approach. We have identified potential inhibitors of Ask1 subunit of a Dam1 complex of C. albicans, which is required to prevent precocious spindle elongation in pre-mitotic phases. The proposed scheme may aid to find virtually potential inhibitors of other unique targets against candida.

Short-Term Pretreatment of Sub-Inhibitory Concentrations of Gentamycin Inhibits the Swarming Motility of Escherichia Coli by Down-Regulating the Succinate Dehydrogenase Gene.

BACKGROUND/AIMS: Motility is a feature of many pathogens that contributes to the migration and dispersion of the infectious agent. Whether gentamycin has a post-antibiotic effect (PAE) on the swarming and swimming motility of Escherichia coli (E. coli) remains unknown. In this study, we aimed to examine whether short-term pretreatment of sub-inhibitory concentrations of gentamycin alter motility of E. coli and the mechanisms involved therein. METHODS: After exposure to sub-inhibitory concentrations (0.8 µg/ml) of gentamicin, the swarming and swimming motility of E. coli was tested in semi-solid media. Real-time PCR was used to detect the gene expression of succinate dehydrogenase (SDH). The production of SDH and fumarate by E. coli pretreated with or without gentamycin was measured. Fumarate was added to swarming agar to determine whether fumarate could restore the swarming motility of E. coli. RESULTS: After pretreatment of E. coli with sub-inhibitory concentrations of gentamycin, swarming motility was repressed in the absence of growth inhibition. The expression of all four subunits of SDH was down-regulated, and the intracellular concentration of SDH and fumarate, produced by E. coli, were both decreased. Supplementary fumarate could restore the swarming motility inhibited by gentamycin. A selective inhibitor of SDH (propanedioic acid) could strongly repress the swarming motility. CONCLUSION: Sub-inhibitory concentrations of gentamycin inhibits the swarming motility of E. coli. This effect is mediated by a reduction in cellular fumarate caused by down-regulation of SDH. Gentamycin may be advantageous for treatment of E. coli infections.

ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis.

Class IA phosphoinositide 3-kinases (PI3Ks) are essential to function of normal and tumor cells, and to modulate immune responses. T lymphocytes express high levels of p110α and p110δ class IA PI3K. Whereas the functioning of PI3K p110δ in immune and autoimmune reactions is well established, the role of p110α is less well understood. Here, a novel dual p110α/δ inhibitor (ETP-46321) and highly specific p110α (A66) or p110δ (IC87114) inhibitors have been compared concerning T cell activation in vitro, as well as the effect on responses to protein antigen and collagen-induced arthritis in vivo. In vitro activation of naive CD4(+) T lymphocytes by anti-CD3 and anti-CD28 was inhibited more effectively by the p110δ inhibitor than by the p110α inhibitor as measured by cytokine secretion (IL-2, IL-10, and IFN-γ), T-bet expression and NFAT activation. In activated CD4(+) T cells re-stimulated through CD3 and ICOS, IC87114 inhibited Akt and Erk activation, and the secretion of IL-2, IL-4, IL-17A, and IFN-γ better than A66. The p110α/δ inhibitor ETP-46321, or p110α plus p110δ inhibitors also inhibited IL-21 secretion by differentiated CD4(+) T follicular (Tfh) or IL-17-producing (Th17) helper cells. In vivo, therapeutic administration of ETP-46321 significantly inhibited responses to protein antigen as well as collagen-induced arthritis, as measured by antigen-specific antibody responses, secretion of IL-10, IL-17A or IFN-γ, or clinical symptoms. Hence, p110α as well as p110δ Class IA PI3Ks are important to immune regulation; inhibition of both subunits may be an effective therapeutic approach in inflammatory autoimmune diseases like rheumatoid arthritis.

Phosphorylation of NR2B NMDA subunits by protein kinase C in arcuate nucleus contributes to inflammatory pain in rats.

The arcuate nucleus (ARC) of the hypothalamus plays a key role in pain processing. Although it is well known that inhibition of NMDA receptor (NMDAR) in ARC attenuates hyperalgesia induced by peripheral inflammation, the underlying mechanism of NMDAR activation in ARC remains unclear. Protein kinase C (PKC) is involved in several signalling cascades activated in physiological and pathological conditions. Therefore, we hypothesised that upregulation of PKC activates NMDARs in the ARC, thus contributing to inflammatory hyperalgesia. Intra-ARC injection of chelerythrine (CC), a specific PKC inhibitor, attenuated complete Freund's adjuvant (CFA) induced thermal and mechanical hyperalgesia in a dose-dependent manner. In vivo extracellular recordings showed that microelectrophoresis of CC or MK-801 (a NMDAR antagonist) significantly reduced the enhancement of spontaneous discharges and pain-evoked discharges of ARC neurons. In addition, CFA injection greatly enhanced the expression of total and phosphorylated PKCγ in the ARC. Interestingly, CFA injection also remarkably elevated the level of phosphorylated NR2B (Tyr1472) without affecting the expression of total NR2B. Importantly, intra-ARC injection of CC reversed the upregulation of phosphorylated NR2B subunits in the ARC. Taken together, peripheral inflammation leads to an activation of NMDARs mediated by PKC activation in the ARC, thus producing thermal and mechanical hyperalgesia.

Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.

Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus.

Neurobehavioral Differences Between Mice Receiving Distinct Neuregulin Variants as Neonates; Impact on Sensitivity to MK-801.

Neuregulin-1 (NRG1) is a well-recognized risk gene for schizophrenia and is often implicated in the neurodevelopmental hypothesis of this illness. Alternative splicing and proteolytic processing of the NRG1 gene produce more than 30 structural variants; however, the neuropathological roles of individual variants remain to be characterized. On the basis of the neurodevelopmental hypothesis of schizophrenia, we administered eNRG1 (0.1~1.0 µg/g), a core epidermal growth factor-like (EGF) domain common for all splicing NRG1 variants, to neonatal mice and compared their behavioral performance with mice challenged with a full mature form of type 1 NRG1 variant. During the neonatal stage, recombinant eNRG1 protein administrated from the periphery passed the blood-brain barrier and activated its receptor (ErbB4) in the brain. In adults, the mice receiving the highest dose exhibited lower locomotor activity and deficits in prepulse inhibition and tonedependent fear learning, although the hearing reduction of the eNRG1-treated mice may explain these behavioral deficits. Neonatal eNRG1 treatment also significantly potentiated MK-801-driven locomotor activity in an eNRG1 dose-dependent manner. In parallel eNRG1 treatment enhanced MK-801-driven c-Fos induction and decreased immunoreactivity for NMDA receptor subunits in adult brain. In contrast, mice that had been treated with the same molar dose of a full mature form of type 1 NRG1 as neonates did not exhibit hypersensitivity to MK-801. However, both animal models exhibited similar hypersensitivity to methamphetamine. Collectively, our findings suggest that aberrant peripheral NRG1 signals during neurodevelopment alter later behavioral traits and auditory functions in the NRG1 subtype-dependent manner.

Probing α4ßδ GABAA receptor heterogeneity: differential regional effects of a functionally selective α4ß1δ/α4ß3δ receptor agonist on tonic and phasic inhibition in rat brain.

In the present study, the orthosteric GABAA receptor (GABAAR) ligand 4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol (Thio-THIP) was found to possess a highly interesting functional profile at recombinant human GABAARs and native rat GABAARs. Whereas Thio-THIP displayed weak antagonist activity at α1,2,5ß2,3γ2S and ρ1 GABAARs and partial agonism at α6ß2,3δ GABAARs expressed in Xenopus oocytes, the pronounced agonism exhibited by the compound at α4ß1δ and α4ß3δ GABAARs was contrasted by its negligible activity at the α4ß2δ subtype. To elucidate to which extent this in vitro profile translated into functionality at native GABAARs, we assessed the effects of 100 µm Thio-THIP at synaptic and extrasynaptic receptors in principal cells of four different brain regions by slice electrophysiology. In concordance with its α6ß2,3δ agonism, Thio-THIP evoked robust currents through extrasynaptic GABAARs in cerebellar granule cells. In contrast, the compound did not elicit significant currents in dentate gyrus granule cells or in striatal medium spiny neurons (MSNs), indicating predominant expression of extrasynaptic α4ß2δ receptors in these cells. Interestingly, Thio-THIP evoked differential degrees of currents in ventrobasal thalamus neurons, a diversity that could arise from differential expression of extrasynaptic α4ßδ subtypes in the cells. Finally, whereas 100 µm Thio-THIP did not affect the synaptic currents in ventrobasal thalamus neurons or striatal MSNs, it reduced the current amplitudes recorded from dentate gyrus granule cells, most likely by targeting perisynaptic α4ßδ receptors expressed at distal dendrites of these cells. Being the first published ligand capable of discriminating between ß2- and ß3-containing receptor subtypes, Thio-THIP could be a valuable tool in explorations of native α4ßδ GABAARs.

New insights in endogenous modulation of ligand-gated ion channels: histamine is an inverse agonist at strychnine sensitive glycine receptors.

Histamine is involved in many physiological functions in the periphery and is an important neurotransmitter in the brain. It acts on metabotropic H1-H4 receptors mediating vasodilatation, bronchoconstriction and stimulation of gastric acid secretion. In the brain histamine is produced by neurons in the tuberomamillary nucleus (TMN), which controls arousal. Histamine is also a positive modulator of the inhibitory Cys-loop ligand-gated ion channel GABAA. We investigated now its effect on the second member of inhibitory Cys-loop ligand-gated ion channels, the strychnine sensitive glycine receptor. We expressed different human and rat glycine receptor subunits in Xenopus laevis oocytes and characterized the effect of histamine using the two electrode voltage clamp technique. Furthermore we investigated native glycine receptors in hypothalamic neurons using the patch-clamp technique. Histamine inhibited α1ß glycine receptors with an IC50 of 5.2±0.3 mM. In presence of 10 mM histamine the glycine dose-response curve was shifted, increasing the EC50 for glycine from 25.5±1.4 µM to 42.4±2.3 µM. In addition, histamine blocked the spontaneous activity of RNA-edited α3ß glycine receptors. Histamine inhibited glycine receptors expressed in hypothalamic TMN neurons with an IC50 of 4.6±0.3 mM. Our results give strong evidence that histamine is acting on the same binding site as glycine, being an inverse agonist that competitively antagonizes glycine receptors. Thus, we revealed histamine as an endogenous modulator of glycine receptors.

The proteasome inhibitor carfilzomib functions independently of p53 to induce cytotoxicity and an atypical NF-κB response in chronic lymphocytic leukemia cells.

PURPOSE: The proteasome consists of chymotrypsin-like (CT-L), trypsin-like, and caspase-like subunits that cleave substrates preferentially by amino acid sequence. Proteasomes mediate degradation of regulatory proteins of the p53, Bcl-2, and nuclear factor-κB (NF-κB) families that are aberrantly active in chronic lymphocytic leukemia (CLL). CLL remains an incurable disease, and new treatments are especially needed in the relapsed/refractory setting. We therefore investigated the effects of the proteasome inhibitor carfilzomib (CFZ) in CLL cells. EXPERIMENTAL DESIGN: Tumor cells from CLL patients were assayed in vitro using immunoblotting, real-time polymerase chain reaction, and electrophoretic mobility shift assays. In addition, a p53 dominant-negative construct was generated in a human B-cell line. RESULTS: Unlike bortezomib, CFZ potently induces apoptosis in CLL patient cells in the presence of human serum. CLL cells have significantly lower basal CT-L activity compared to normal B and T cells, although activity is inhibited similarly in T cells versus CLL. Co-culture of CLL cells on stroma protected from CFZ-mediated cytotoxicity; however, PI3K inhibition significantly diminished this stromal protection. CFZ-mediated cytotoxicity in leukemic B cells is caspase-dependent and occurs irrespective of p53 status. In CLL cells, CFZ promotes atypical activation of NF-κB evidenced by loss of cytoplasmic IκBα, phosphorylation of IκBα, and increased p50/p65 DNA binding, without subsequent increases in canonical NF-κB target gene transcription. CONCLUSIONS: Together, these data provide new mechanistic insights into the activity of CFZ in CLL and support phase I investigation of CFZ in this disease.

Effects of continuous ingestion of hesperidin and glucosyl hesperidin on vascular gene expression in spontaneously hypertensive rats.

Hesperidin (HES) and glucosyl hesperidin (GHES) have antihypertensive effects. In the present study, to clarify the antihypertensive mechanisms, we compared the effects of continuous ingestion of HES and GHES in spontaneously hypertensive rats (SHRs). HES and GHES ingestion for 8 wk significantly prevented hypertension and suppressed the mRNA expression of NADPH oxidase subunits and thromboxane A2 synthase in SHR aortas. Further, hesperetin, a common metabolite of HES and GHES, reduced thromboxane B2 release from SHR aortas. These findings indicate that continuous ingestion of HES and GHES prevents hypertension via regulating the gene expression related to the modulation of vascular tone.

Na⁺-K⁺-ATPase, a potent neuroprotective modulator against Alzheimer disease.

Alzheimer disease (AD) is a neurodegenerative disorder clinically characterized by progressive cognitive and memory dysfunction, which is the most common form of dementia. Although the pathogenesis of neuronal injury in AD is not clear, recent evidences suggest that Na⁺-K⁺-ATPase plays an important role in AD, and may be a potent neuroprotective modulator against AD. This review aims to provide readers with an in-depth understanding of Na⁺-K⁺-ATPase in AD through these modulations of some factors that are as follows, which leads to the change of learning and memory in the process of AD. 1. The deficiency in Na⁺, K⁺-ATPase α1, α2 and α3 isoform genes induced learning and memory deficits, and α isoform was evidently changed in AD, revealing that Na⁺, K⁺-ATPase α isoform genes may play an important role in AD. 2. Some factors, such as ß-amyloid, cholinergic and oxidative stress, can modulate learning and memory in AD through the mondulation of Na⁺-K⁺-ATPase activity. 3. Some substances, such as Zn, s-Ethyl cysteine, s-propyl cysteine, citicoline, rivastigmine, Vit E, memantine, tea polyphenol, curcumin, caffeine, Alpinia galanga (L.) fractions, and Bacopa monnieri could play a role in improving memory performance and exert protective effects against AD by increasing expression or activity of Na⁺, K⁺-ATPase.

Characterization of PI3K class IA isoforms with regulatory subunit p55alpha using a scintillation proximity assay.

Differential activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway has been linked to cancer. Activation occurs through gene amplification and activating mutations. High-frequency mutations in the gene encoding the p110alpha catalytic subunit of PI3K (PIK3CA) have been observed in a variety of tumors including colon, brain, breast, ovarian, and gastric. Inhibition of PI3K kinase activity may provide a specific way to treat multiple types of human cancer. A scintillation proximity assay (SPA) was developed to detect phosphatidylinositol 3-kinase catalytic activity. Using this assay format, steady-state kinetic parameters were compared for the PI3K class IA enzymes p110alpha, p110beta, and p110delta, each coexpressed with the regulatory subunit p85alpha or splice variant p55alpha. Inhibition by the natural product wortmannin and LY294002 was detected with potencies consistent with alternate assay formats. Other biochemical assay formats have been described for phosphoinositide 3-kinases but each has its unique limitations. The simple, inexpensive, sensitive high-throughput nature of the SPA format has advanced our knowledge of isoform-specific enzymology and will facilitate the discovery of novel PI3K inhibitors.

Pharmacological characterization of ligands at recombinant NMDA receptor subtypes by electrophysiological recordings and intracellular calcium measurements.

Generation of in vitro cellular assays using fluorescence measurements at heterologously expressed NMDA receptors would speed up the process of ligand characterization and enable high-throughput screening. The major drawback to the development of such assays is the cytotoxicity caused by Ca(2+)-flux into the cell via NMDA receptors upon prolonged activation by agonists present in the culture medium. In the present study, we established four cell lines with stable expression of NMDA receptor subtypes NR1/NR2A, NR1/NR2B, NR1/NR2C, or NR1/NR2D in BHK-21 cells. To assess the usefulness of the stable cell lines in conjunction with intracellular calcium ([Ca(2+)](i)) measurements for evaluation of NMDA receptor pharmacology, several ligands were characterized using this method. The results were compared to parallel data obtained by electrophysiological recordings at NMDA receptors expressed in Xenopus oocytes. This comparison showed that agonist potencies determined by [Ca(2+)](i) measurements and electrophysiological recordings correlated well, meaning that the stable cell lines in conjunction with [Ca(2+)](i) measurements provide a useful tool for characterization of NMDA receptor ligands. The agonist series of conformationally constrained glutamate analogues (2S,3R,4S)-alpha-(carboxycyclopropyl)glycine (CCG), 1-aminocyclobutane-r-1,cis-3-dicarboxylic acid (trans-ACBD), and (+/-)-1-aminocyclopentane-r-1,cis-3-dicarboxylic acid (cis-ACPD), as well as the highly potent agonist tetrazolylglycine were among the characterized ligands that were assessed with respect to subtype selectivity at NMDA receptors. However, none of the characterized agonists displays more than 2-3 fold selectivity towards a specific NMDA receptor subtype. Thus, the present study provides a broad pharmacological characterization of structurally diverse ligands at recombinant NMDA receptor subtypes.

Partial deletion of the nicotinic cholinergic receptor alpha 4 or beta 2 subunit genes changes the acetylcholine sensitivity of receptor-mediated 86Rb+ efflux in cortex and thalamus and alters relative expression of alpha 4 and beta 2 subunits.

Alpha4 and beta2 nicotinic cholinergic receptor (nAChR) subunits can assemble in heterologous expression systems as pentameric receptors with different subunit stoichiometries that exhibit differential sensitivity to activation by acetylcholine, yielding biphasic concentration-effect curves. nAChR-mediated (86)Rb(+) efflux in mouse brain synaptosomes also displays biphasic acetylcholine (ACh) concentration-response curves. Both phases are mediated primarily by alpha4beta2(*)-nAChR, because deletion of either the alpha4 or beta2 subunit reduces response at least 90%. A relatively larger decrease in the component of (86)Rb(+) efflux with lower ACh sensitivity occurred with partial deletion of alpha4 (alpha4(+/-)), whereas a larger decrease in the component with higher ACh sensitivity was elicited by partial deletion of beta2 (beta2(+/-)). Immunoprecipitation with selective antibodies demonstrated that more than 70% of [(3)H]epibatidine binding sites in both regions contained only alpha4 and beta2 subunits. Subsequently, alpha4 and beta2 subunit content in the cortex and thalamus of alpha4 and beta2 wild types and heterozygotes was analyzed with Western blots. Partial deletion of alpha4 decreased and partial deletion of beta2 increased the relative proportion of the alpha4 subunit in assembled receptors. Although these methods do not allow exact identification of stoichiometry of the subtypes present in wild-type cortex and thalamus, they do demonstrate that cortical and thalamic nAChRs of the alpha4(+/-) and beta2(+/-) genotypes differ in relative expression of alpha4 and beta2 subunits a result that corresponds to the relative functional changes observed after partial gene deletion. These results strongly suggest that alpha4beta2-nAChR with different stoichiometry are expressed in native tissue.

Homogenous time resolved fluorescence assay to identify modulators of cap-dependent translation initiation.

Eukaryotic initiation factor (eIF) 4F plays a key role in recruiting 40S ribosomes and associated factors to mRNA templates during translation initiation. The function of this heterotrimeric complex is to deliver an RNA helicase to the 5' cap proximal region of mRNAs in preparation for ribosome binding. To study the interaction between subunits of this complex, as well as identify small molecules that could interfere with their association, we developed a time resolved fluorescence assay that allows monitoring of interactions between two subunits of eIF4F. We have performed a small molecule chemical screen of >73,000 compounds using this assay.

Methylation of green tea polyphenols affects their binding to and inhibitory poses of the proteasome beta5 subunit.

Previously, we showed that ester carbon-containing tea polyphenols, including (-)-epigallocatechin gallate [(-)-EGCG] and (-)-epicatechin-3-gallate [(-)-ECG], potently inhibit proteasomal chymotrypsin-like activity. In addition, our in silico docking study suggested that a particular pose of (-)-EGCG could lead to potential covalent modification of the N-terminal threonine (Thr 1) of the proteasome beta5 subunit in the chymotrypsin-like active site. It has been suggested that some major biotransformation reactions, such as methylation, could result in reduced biological activity of (-)-EGCG in vivo. We hypothesize that methylation reduces binding of (-)-EGCG to the beta5 subunit of the proteasome and, therefore, decreases its proteasomal chymotrypsin-like-inhibitory potency. Here, we report that, while methylation has no effect on nucleophilic susceptibility of (-)-EGCG and (-)-ECG, it may disrupt the ability of these polyphenols to interact with Thr 1 of the proteasome beta5 subunit. In silico docking shows that methylation results in the tea polyphenols' ester carbon being moved away or blocked entirely from Thr 1. Additionally, methylation impairs the ability of (-)-EGCG and (-)-ECG to dock in a consistent low energy pose. These observations, no change in nucleophilic susceptibility, moving or blocking the ester carbon from Thr 1, and lack of a consistent docking pose, suggest that methylation disrupts the ability of (-)-EGCG and (-)-ECG to bind to the proteasome beta5 subunit, which may then diminish their proteasomal chymotrypsin-inhibitory and, therefore, other biological activities.