Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.

Selective inhibitors of CYP24A1 represent an important synthetic target in a search for novel vitamin D compounds of therapeutic value. In the present work, we show the synthesis and biological properties of two novel side chain modified 2-methylene-19-nor-1,25(OH)(2)D(3) analogs, the 22-imidazole-1-yl derivative 2 (VIMI) and the 25-N-cyclopropylamine compound 3 (CPA1), which were efficiently prepared in convergent syntheses utilizing the Lythgoe type Horner-Wittig olefination reaction. When tested in a cell-free assay, both compounds were found to be potent competitive inhibitors of CYP24A1, with the cyclopropylamine analog 3 exhibiting an 80-1 selective inhibition of CYP24A1 over CYP27B1. Addition of 3 to a mouse osteoblast culture sustained the level of 1,25(OH)(2)D(3), further demonstrating its effectiveness in CYP24A1 inhibition. Importantly, the in vitro effects on human promyeloid leukemia (HL-60) cell differentiation by 3 were nearly identical to those of 1,25(OH)(2)D(3) and in vivo the compound showed low calcemic activity. Finally, the results of preliminary theoretical studies provide useful insights to rationalize the ability of analog 3 to selectively inhibit the cytochrome P450 isoform CYP24A1.

We herein report a material technique to control the shapes of cell nuclei by the design of the microtopography of substrates to which the cells adhere. Poly(D,L-lactide-co-glycolide) (PLGA) micropillars or micropits of a series of height or depth were fabricated, and some surprising self deformation of the nuclei of bone marrow stromal cells (BMSCs) was found in the case of micropillars with a sufficient height. Despite severe nucleus deformation, BMSCs kept the ability of proliferation and differentiation. We further demonstrated that the shapes of cell nuclei could be regulated by the appropriate micropillar patterns. Besides circular and elliptoid shapes, some unusual nucleus shapes of BMSCs have been achieved, such as square, cross, dumbbell, and asymmetric sphere-protrusion.

The release of reactive oxygen species (ROS) as side products of aerobic metabolism in the mitochondria is an unavoidable consequence. As the capacity of organisms to deal with this exposure declines with age, accumulation of molecular damage caused by ROS has been defined as one of the central events during the ageing process in biological systems as well as in numerous diseases such as Alzheimer's and Parkinson's Dementia. In the filamentous fungus Podospora anserina, an ageing model with a clear defined mitochondrial etiology of ageing, in addition to the mitochondrial aconitase the ATP synthase alpha subunit was defined recently as a hot spot for oxidative modifications induced by ROS. In this report we show, that this reactivity is not randomly distributed over the ATP Synthase, but is channeled to a single tryptophan residue 503. This residue serves as an intra-molecular quencher for oxidative species and might also be involved in the metabolic perception of oxidative stress or regulation of enzyme activity. A putative metal binding site in the proximity of this tryptophan residue appears to be crucial for the molecular mechanism for the selective targeting of oxidative damage.

Antagonists of L-type Ca²⁺ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C⁻/⁺ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C⁻/⁺ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C⁻/⁺ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C⁺/⁺ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca²⁺ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease.

Cryopreservation is an essential technique in basic research and clinical applications of human embryonic stem (hES) cells. Cryopreserved hES cells are fragile and undergo post-thaw apoptosis. We performed gene pathway analysis on cryopreserved and thawed hES cells to examine the effect of Y-27632, a Rho-associated kinase (ROCK) inhibitor, on apoptosis and associated molecular events. Y-27632 was added to the cryopreservation solution and/or the post-thaw medium of two hES cell lines (KhES-1, KhES-3). Post-thaw apoptosis was recorded as a function of time using Giemsa staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Apoptosis plateaued 12h after the untreated hES cells were thawed. Gene pathway analysis showed the activation of IL-1β, TGF-β, and their respective receptors (IL-1R, ACVR1C) in the mitogen-activated protein kinase (MAPK) pathway, which resulted in the upregulation of caspase-8 and -10. Quantitative RT-PCR confirmed the upregulation of IL-1β, TGF-β, their respective receptors, and caspase-10 and -3. As these molecules were suppressed by Y-27632, gene pathways involving these molecules probably depend on ROCK activation. The TGF-β receptor antagonist, SB-431542, and an inhibitor of p38MAPK, SB-203580, did not affect apoptosis. Combining Y-27632 with SB-203580, however, resulted in an increase in the survival rate compared with the control. This suggests that the initiation of apoptosis depends on cytokine interactions and multiple ways exist to reduce post-thaw apoptosis in hES cells. Y-27632 can suppress cytokine interactions and the MAPK pathway, thereby reducing the occurrence of apoptosis, and is an effective cryoprotectant for hES cells.

Estradiol affects hippocampal-dependent spatial memory and underlying structural and electrical synaptic plasticity in female mice and rats. Using estrogen receptor (ER) alpha and beta knockout mice and wild-type littermates, we investigated the role of ERs in estradiol effects on multiple pathways important for hippocampal plasticity and learning. Six hours of estradiol administration increased immunoreactivity for phosphorylated Akt throughout the hippocampal formation, whereas 48 h of estradiol increased immunoreactivity for phosphorylated TrkB receptor. Estradiol effects on phosphorylated Akt and TrkB immunoreactivities were abolished in ER alpha and ER beta knockout mice. Estradiol also had distinct effects on immunoreactivity for post-synaptic density 95 (PSD-95) and brain derived-neurotrophic factor (BDNF) mRNA in ER alpha and beta knockout mice. Thus, estradiol acts through both ERs alpha and beta in several subregions of the hippocampal formation. The different effects of estradiol at 6 and 48 h indicate that several mechanisms of estrogen receptor signaling contribute to this female hormone's influence on hippocampal synaptic plasticity. By further delineating these mechanisms, we will better understand and predict the effects of endogenous and exogenous ovarian steroids on mood, cognition, and other hippocampal-dependent behaviors.

The serotonergic centrifugal system innervating the main olfactory bulb (MOB) plays a key role in the modulation of olfactory processing. We have previously demonstrated that this system suffers adaptive changes under conditions of a lack of olfactory input. The present work examines the response of this centrifugal system after mitral cell loss in the Purkinje cell degeneration (pcd) mutant mice. The distribution and density of serotonergic centrifugal axons were studied in the MOB of control and pcd mice, both before and after the loss of mitral cells, using serotonin (5-HT) and 5-HT transporter immunohistochemistry. Studies of the amount of 5-HT and its metabolite, 5-hydroxyindole acetic acid (5-HIAA), were performed by means of high-performance liquid chromatography (HPLC), and the relative amounts of brain-derived neurotrophin factor, BDNF, and its major receptor, tropomyosin-related kinase B (TrkB), were measured by Western blot. Our study revealed that the serotonergic system develops adaptive changes after, but not before, mitral cell loss. The lack of the main bulbar projection cells causes a decrease in the serotonergic input received by the MOB, whereas the number of serotonergic cells in the raphe nuclei remains constant. In addition, one of the molecules directly involved in serotonergic sprouting, the neurotrophin BDNF and its main receptor TrkB, underwent alterations in the MOBs of the pcd animals even before the loss of mitral cells. These data indicate that serotonergic function in the MOB is closely related to olfactory activity and that mitral cell loss induces serotonergic plastic responses.

Cisplatin-based chemotherapy regimens are preferred in the treatment of a variety of cancers. The present study investigated early cumulative molecular effects of therapeutic dose-levels of bleomycin, etoposide and cisplatin (BEP) in the testis and their modulation by an antioxidant cocktail (AO). Adult male Sprague-Dawley rats (N=7/group [G]) were treated with BEP as follows: G1 - control; G2 - AO (α-tocopherol [100 mg/kg], l-ascorbic acid [50 mg/kg], Zn [40 mg/l] and Se [100 μg/l]); G3 - B, 1.5 mg/kg on day 2; E, 15 mg/kg and P, 3 mg/kg for 4 days, and G4 - similar to G3 but also treated with AO for 4 days. In G3, the testis weight, sperm count and motility, and activities of enzymatic antioxidants decreased and lipid peroxidation increased compared to that in G1 (P<0.05). Seminiferous epithelial sloughing and degeneration were observed. In G3, mRNA levels of p53, Bcl-2 and Bax were unaltered but protein expression of p53 and Bax was up-regulated and that of Bcl-2 was down-regulated (P<0.05). These changes led to an increase in terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) positive germ cells indicating cell death (P<0.05). The AO recovered the BEP-induced molecular alterations to control levels. The mechanism of BEP-induced early testicular damage involves the initiation of oxidative stress, up-regulation of pro-apoptotic proteins and induction of cell death. Further, the induced testicular structural changes are negligible and less than those observed in single drug exposure studies reported in literature. The AO significantly ameliorates the BEP-induced pathogenesis of testicular damage suggesting its potential therapeutic uses.

The purpose of this study was to assess the feasibility of initiating primary cultures of corneal endothelial cells from patients suffering from Fuchs endothelial corneal dystrophy (FECD; MIM# 1036800). We also evaluated which conditions yielded the best results for culture. Twenty-nine patients undergoing Descemet stripping automated endothelial keratoplasty consented to the use of their excised Descemet's membrane for this study. Out of the 29 specimens, 18 successfully initiated a culture. Cell morphology varied between endothelial (rounded, slightly elongated cells, n = 12) and fibroblastic-like (thin and very elongated cells, n = 6). These differences in cell morphology were also observed with the normal human corneal endothelial cell cultures. The cultures that initially presented an endothelial morphology maintained their shape in subcultures. Clusterin expression was similar in FECD and normal endothelial cells. Transmission electron microscopy of FECD Descemet's membranes showed a high degree of various abnormalities generally found in this disease, such as a thickened Descemet's membrane, presence of a posterior banded layer, presence of a fibrillar layer and striated bodies of various sizes and periodicities. Patient's age was predictive of culture success, all younger FECD donors generating cultures of endothelial morphology. The absence of a fibrillar layer was also a factor associated with greater success. Culture success was not dependent on specimen size, specimen pigmentation, or patient's preoperative central corneal thickness. In conclusion, this paper shows for the first time that central Descemet's membranes of patients suffering from FECD possess proliferative endothelial cells that can be isolated and cultured without viral transduction, opening the way for new in vitro studies of this disease.

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects embryo development, implantation and fertility in humans. The underlying molecular mechanism by which TCDD suppresses implantation remains largely unknown. We used the trophoblastic spheroids (embryo surrogate)-endometrial cells co-culture assay to study the attachment of trophoblastic spheroids (BeWo and Jeg-3) onto the endometrial epithelial (RL95-2 and Ishikawa) cells. TCDD dose-dependently induced cytochrome P450 1A1 (Cyp1A1) expression in trophoblastic and endometrial epithelial cells. Moreover, TCDD at 1 and 10 nM suppressed β-catenin (a Wnt-signaling molecule) and E-cadherin expression, as well as spheroids attachment onto endometrial cells. Interestingly, activation of the canonical Wnt-signaling pathway via Wnt3a or lithium chloride reverted the suppressive effect of TCDD on β-catenin and E-cadherin expressions in the BeWo and RL95-2 cells, and restored the spheroids attachment rate to be comparable to the untreated controls. Taken together, TCDD induces Cyp1A1 expression, modulates the Wnt-signaling pathway and suppresses spheroids attachment onto endometrial cells.

Potential metabolites from the metabolic pathways could be therapeutic targets and useful for the discovery of broad spectrum drugs. UPLC/ESI-SYNAPT-HDMS coupled with pattern recognition methods including PCA, PLS-DA, OPLS-DA and Heatmap were integrated to examine the global metabolic signature of insomnia and intervention effects of Jujuboside A (JuA). Six unique pathways of the insomnia were identified using Ingenuity Pathway Analysis (IPA) software. The VIP-value threshold cutoff of the metabolites was set to 10, above this threshold, were filtered out as potential target biomarkers. Sixteen distinct metabolites were identified from these pathways, and 6 of them can be considered for rational drug design. It was further experimental validation that the changes in metabolic profiling were restored to their baseline values after JuA treatment according to the multivariate data analysis. Potential metabolite network of the insomnia was preliminarily predicted JuA-target interaction networks, and could be further explored for in silico docking studies with suitable drugs. Thus, our method is an efficient procedure for drug target identification through metabolic analysis. It can guide testable predictions, provide insights into drug action mechanisms and enable us to increase research productivity toward metabolomic drug discovery.

Fusion systems are known to increase the expression of difficult to express recombinant proteins in soluble form to facilitate their purification. Rabies glycoprotein was also tough to express at sufficient level in soluble form in both E. coli and plant. The present work was aimed to over-express and purify this membrane protein from soluble extract of E. coli. Fusion of Small Ubiqutin like Modifier (SUMO) with rabies glycoprotein increased ~1.5 fold higher expression and ~3.0 fold solubility in comparison to non-fused in E. coli. The SUMO fusion also simplified the purification process. Previously engineered rabies glycoprotein gene in tobacco plants provides complete protection to mice, but the expression was very low for purification. Our finding demonstrated that the SUMO-fusion was useful for enhancing expression and solubility of the membrane protein and again proves to be a good alternative technology for applications in biomedical and pharmaceutical research.

Among markers of glioblastoma initiating cells, AC133 has been shown to be associated with glioblastoma resistance and malignancy. Recently, it was demonstrated that increasing oxygen tension (pO2) down-regulated AC133 expression in glioblastoma cells in vitro. In order to better understand extrinsic factor regulation of AC133, this work aimed to investigate the relationship between cell culture pO2, AC133 expression, and tumor development and phenotype. Using treatments with CoCl2 and HIF-1α shRNA knockdowns on non-sorted human primary glioblastoma cells cultured at low (3%) versus high (21%) oxygen tension, we established a responsibility for low pO2 in the maintenance of high levels of AC133 expression, with a major but non-exclusive role for HIF-1α. We also demonstrated that human glioblastoma cells previously cultured under high oxygen tension can lose part of their aggressiveness when orthotopically engrafted in SCID mice or lead to tumors with distinct phenotypes and no re-expression of AC133. These observations showed that the specific pO2 microenvironment irreversibly impacts glioblastoma cell phenotypes, highlighting the pertinence of culture conditions when extrapolating data from xenogenic models to human cells in their source environment. They also raised AC133 as a marker of non-exposure to oxygenated areas rather than a marker of aggressiveness or low pO2 niches.

Pancreatic cancer is a complex malignancy arising from the accumulation of genetic and epigenetic defects in the affected cells. Standard chemotherapy for patients with advanced disease shows only modest effects and is associated with considerable toxicity. Overexpression or aberrant activation of members of the epidermal growth factor receptor tyrosine kinase family, which includes EGFR and HER-2, occurs frequently and is associated with multiple drug resistance and decreased patient survival. In this study, we have investigated the therapeutic potential of AS104, a novel compound of the triazene class, with potential inhibitory effects on EGFR. We found that treatment of cells with AS104 causes significant reduction of cell growth and metabolic activity in four human pancreatic cancer cell lines. Furthermore, we show that the AS104-mediated induction of apoptotic cell death is associated with stimulation of autophagy in a dose-dependent manner. Treatment of cells with AS104 results in significant down-regulation of EGFR and HER-2 expression and activity and subsequent inhibition of downstream signaling proteins. Quantitative RT-PCR analysis and assays with proteasome inhibitors revealed that AS104 regulates the expression of EGFR and HER-2 at the transcriptional level. These findings provide for the first time experimental evidence for efficacy of AS104 in the simultaneous transcriptional repression of EGFR and HER-2 genes and suggest that AS104 may have therapeutic potential in the treatment of pancreatic cancers that express high levels of the aforementioned receptor tyrosine kinases.

The statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have been proven to be effective in lowering cholesterol and as anti-lipid agents against cardiovascular disease. Recent reports demonstrate an anticancer effect induced by the statins through inhibition of cell proliferation. Probably, these effects are due to suppression of the mevalonate pathway leading to the depletion of various downstream products that play an essential role in cell cycle progression, cell signaling and membrane integrity. To date, although many hypotheses have been proposed, the exact mechanism at the basis of cancer cell growth arrest induced by statins is not known. In this study, we have demonstrated that simvastatin, at a dose of 20 µM for 24-72 h, induced in cancer cells but not in normal cells precise features of apoptosis including increased DNA fragmentation while, at the molecular level simvastatin induced overexpression of the pro-apoptotic gene Bax together with an inhibition of BCL-2, the gene that has the well-known function of protecting cells from apoptosis. The simvastatin-mediated induction of apoptosis in similar cancer cells but not in normal cells is very interesting and may be at the basis of cancer therapy using statins, usually in combination with chemotherapy or to be used as a cancer protective drug. Simvastatin may, thus, play a dual prophylactic role as a lipid-lowering drug for the prevention of heart disease and as an anticancer agent to prevent certain types of cancers.

Rhea (http://www.ebi.ac.uk/rhea) is a comprehensive resource of expert-curated biochemical reactions. Rhea provides a non-redundant set of chemical transformations for use in a broad spectrum of applications, including metabolic network reconstruction and pathway inference. Rhea includes enzyme-catalyzed reactions (covering the IUBMB Enzyme Nomenclature list), transport reactions and spontaneously occurring reactions. Rhea reactions are described using chemical species from the Chemical Entities of Biological Interest ontology (ChEBI) and are stoichiometrically balanced for mass and charge. They are extensively manually curated with links to source literature and other public resources on metabolism including enzyme and pathway databases. This cross-referencing facilitates the mapping and reconciliation of common reactions and compounds between distinct resources, which is a common first step in the reconstruction of genome scale metabolic networks and models.

As the relevant literature and the number of experiments increase at a super linear rate, databases that curate and collect experimentally verified microRNA (miRNA) targets have gradually emerged. These databases attempt to provide efficient access to this wealth of experimental data, which is scattered in thousands of manuscripts. Aim of TarBase 6.0 (http://www.microrna.gr/tarbase) is to face this challenge by providing a significant increase of available miRNA targets derived from all contemporary experimental techniques (gene specific and high-throughput), while incorporating a powerful set of tools in a user-friendly interface. TarBase 6.0 hosts detailed information for each miRNA-gene interaction, ranging from miRNA- and gene-related facts to information specific to their interaction, the experimental validation methodologies and their outcomes. All database entries are enriched with function-related data, as well as general information derived from external databases such as UniProt, Ensembl and RefSeq. DIANA microT miRNA target prediction scores and the relevant prediction details are available for each interaction. TarBase 6.0 hosts the largest collection of manually curated experimentally validated miRNA-gene interactions (more than 65,000 targets), presenting a 16.5-175-fold increase over other available manually curated databases.

With the growing number of experimentally resolved structures of macromolecular complexes, it becomes clear that the interactions that involve protein structures are mediated not only by the protein domains, but also by various non-structured regions, such as interdomain linkers, or terminal sequences. Here, we present DOMMINO (http://dommino.org), a comprehensive database of macromolecular interactions that includes the interactions between protein domains, interdomain linkers, N- and C-terminal regions and protein peptides. The database complements SCOP domain annotations with domain predictions by SUPERFAMILY and is automatically updated every week. The database interface is designed to provide the user with a three-stage pipeline to study macromolecular interactions: (i) a flexible search that can include a PDB ID, type of interaction, SCOP family of interacting proteins, organism name, interaction keyword and a minimal threshold on the number of contact pairs; (ii) visualization of subunit interaction network, where the user can investigate the types of interactions within a macromolecular assembly; and (iii) visualization of an interface structure between any pair of the interacting subunits, where the user can highlight several different types of residues within the interfaces as well as study the structure of the corresponding binary complex of subunits.

Objective. To investigate the indication and limitations of uterine artery embolization (UAE), we retrospectively analyzed the case of patients with uterine leiomyomas who had undergone UAE. Methods. During the past 7 years, 25 patients with uterine leiomyomas had undergone UAE in our hospital. UAE was indicated in patients with menstrual disturbances such as hypermenorrhea or dysmenorrhea. The outcomes of this procedure for uterine leiomyomas were analyzed. Results. Improvement in the menstrual symptoms and/or reduction in the leiomyoma size after UAE were observed in 24 patients (96.0%). There was mean 67.9% reduction in the volume of leiomyomas at six months after UAE (P < 0.05). However, the symptoms recurred after UAE in 6 patients (24.0%), with multiple or intramural leiomyomas larger than 7 cm, which necessitated additional procedures. Conclusion. The indications and limitations of the UAE should be considered because of its noneffectiveness and/or recurrence in over 20% of patients with uterine leiomyomas.