Human genome–guided identification of memory-modulating drugs

In the last decade there has been an exponential increase in knowledge about the genetic basis of complex human traits, including neuropsychiatric disorders. It is not clear, however, to what extent this knowledge can be used as a starting point for drug identification, one of the central hopes of the human genome project. The aim of the present study was to identify memory-modulating compounds through the use of human genetic information. We performed a multinational collaborative study, which included assessment of aversive memory—a trait central to posttraumatic stress disorder—and a gene-set analysis in healthy individuals. We identified 20 potential drug target genes in two genomewide-corrected gene sets: the neuroactive ligand–receptor interaction and the long-term depression gene set. In a subsequent double-blind, placebo-controlled study in healthy volunteers, we aimed at providing a proof of concept for the genome-guided identification of memory modulating compounds. Pharmacological intervention at the neuroactive ligand–receptor interaction gene set led to significant reduction of aversive memory. The findings demonstrate that genome information, along with appropriate data mining methodology, can be used as a starting point for the identification of memory-modulating compounds.Recent advances in human genetics have led to an unprecedented rate of discovery of genes related to complex human disease, including neuropsychiatric disorders (1–3). The human genome–based gain of knowledge is certainly expected to have a large impact on drug discovery in complex human disease (4–6). It is, however, still not clear to what extent this knowledge can be used as a starting point for the identification of druggable molecular pathways of complex traits (7), including mental disorders (8).Genomewide association studies (GWASs) using single-marker statistics have been very successful in identifying trait-associated single-gene loci (9). It is, however, widely accepted that single marker–based analyses have limited power to identify the genetic basis of a given trait, as for example, many loci will fail to reach stringent genomewide significance threshold, despite the fact that they may be genuinely associated with the trait. Triggered by statistical approaches for the analysis of gene expression, gene set–based analytical methods have recently become available. These methods aim at identifying biologically meaningful sets of genes associated with a certain trait, rather than focusing on a single GWAS gene locus (10). By taking into account prior biological knowledge, gene set–based approaches examine whether test statistics for a group of related genes have consistent deviation from chance (10). As shown recently in studies on autism (11), bipolar disorder (12, 13), attention deficit hyperactivity disorder (ADHD) (14), and schizophrenia (15), such approaches can convincingly identify convergent molecular pathways relevant to neuropsychiatry. Importantly, the identification of groups of functionally related genes is likely to facilitate drug discovery, because the most significant single loci from a GWAS might not be the best candidates for therapeutic intervention (7, 10).In the present study, we focused on emotionally aversive memory—a trait central to anxiety disorders such as posttraumatic stress disorder (PTSD) (16–23). Strong memory for emotionally arousing events can be seen as a primarily adaptive phenomenon, which helps us to remember vital information (e.g., dangerous situations). In case of an extremely aversive event, however, this mechanism can also lead to intrusive and persistent traumatic memories, thereby contributing to the development and symptoms of PTSD (18–22). Symptoms related to aversive memory include intrusive daytime recollections, traumatic nightmares, and flashbacks in which components of the event are relived. Aversive memory is a genetically complex trait as shown both in healthy subjects and in traumatized individuals (17, 23). Furthermore, we recently reported evidence suggesting a genetic link between the predisposition to build strong aversive memories and the risk for PTSD (16).Based on these observations, we developed a process (Fig. 1) aimed at identifying gene sets related to human aversive memory, followed by a pharmacological intervention study as proof-of-concept for the genome-guided identification of memory-modulating drugs.Open in a separate windowFig. 1.Drug discovery process.     

Filamentous-actins in human hepatocarcinoma cells with CLSM

AIM:To establish a method for optical sections of HepG2human hepatoblastoma cells with confocal laser scanningmicroscope (CLSM) and to study the spatial structure offilamentous actin (F-actin) in HepG2 cells.METHODS:HepG2 cells were stained with FITC-phalloidinthat specifically binds F-actin,with propidium iodide (PI) tothe nucleus,and scanned with a CLSM to generate opticallysectioned images.A series of optical sections takensuccessively at different focal levels in steps of 0.7μm werereconstructed with the CLSM reconstruction program.RESULTS:CLSM images showed that the FITC-stained F-actin was abundant microfilament bundles parallel or nettedthrough the whole cell and its processes.Most F-actinmicrofilaments extended through the cell from one part towardthe other or run through the process.Some microfilamentswere attached to the plasma membrane,or formed astructural bridge connecting to the neighboring cells.CONCLUSION:A method for double labeling HepG2 humanhepatoblastoma cells and CLSM imaging F-actin microfilamentsand nuclei by image thin optical sections and spatial structurewas developed.It provides a very useful way to study thespatial structure of F-actin.     

The αβδ of ion channels in human islet cells

Ion channels in the plasma membrane are critical for coupling metabolic signals to hormone release from pancreatic islet cells. The increased availability of human islets for research purposes has recently enabled us to perform a more detailed characterization of ion channels in human β-cells as well as in human δ- and α-cells. This addendum summarizes the major differences between mouse and human β-cells and discusses some of the remaining open questions. In addition, differences between the ion channel complements of human β-cells and islet non-β-cells are addressed.     

Multidrug resistance reversal in human gastric carcinoma cells by neferine

AIM:To investigate the reversal effect of neferine on multidrugresistance in human gastric carcinoma cell line.METHODS:Cells of a human gastric cancer cells line,SGC7901,and its vincristine(VCR)-resistant variant,SGC7901/VCR,were cultivated with or without neferine and/or VCR.Thecytotoxic effect of VCR was evaluated by the MTT assay.Cellapoptosis induced by VCR was determined by flow cytometry(FCM).The expression of P-glycoprotein(P-gp)and amultidrug-resistance-associated protein(MRP)in cells wasexamined by immunofluorescence and FCM.RESULTS:Neferine at the concentration from 2.5 μmol/L to10 μmol/L had no cytotoxidty to SGC7901 cells,and its variantSGC7901/VCR cells.The IC_(50) of VCR against SGC7901 andSGC7901/VCR cells was 0.059 μg/mL and 2.32 μg/mL,respectively,indicating that SGC7901/VCR cells were 39 timesmore resistant to VCR than its parent SGC7 901 cells.Altertreatment with neferine at concentrations of 2.5,5 and10 μmol/L,the IC_(50) of VCR to SGC7901/VCR cell linedecreased to 0.340,0.128 and 0.053 μg/mL,respectively,thus,increased the chemosensitivity by 6.8-,18.1-and43.8-fold,respectively.SGC7901/VCR cells were apoptosisresistant to VCR.Neferine(2.5,5 and 10 μmol/L)promotedthe VCR-induced apoptosis of SGC7901/VCR cells in a dose-dependent manner.The expressions of P-gp and MRP werestrongly positive in SGC7901/VCR cells,which were significantlydown-regulated alter treatment with neferine(10 μmol/L)for 24 h.CONCLUSION:Neferine reverses multidrug resistance ofhuman gastric carcinoma SGC7901/VCR cells,which maybe associated with the down-regulations of P-gp and MRPexpression in SGC701/VCR cells.     

Glycoxidation of low–density lipoprotein promotes multiple apoptotic pathways and NFkB activation in human coronary cells

Apoptosis of arterial cells induced by oxidized low–density lipoprotein (oxLDL) is thought to contribute to the progression of vascular dysfunction and atherogenesis. It is well established that diabetes mellitus is accompanied by both glycosylation and oxidation LDL, but the biological effects of these modified lipoproteins are poorly understood. We demonstrate here that glycosylated oxLDL (glc–oxLDL) promotes apoptotic signaling in human coronary smooth muscle cells. This was associated by a decrease of the antiapoptotic protein Bcl–2, an increase of the pro–apoptotic protein Bax, and activation of caspase 3. Glc–oxLDL also activated NFkB and decreased IkB, these effects were more pronounced than those achieved with oxLDL. Our study shows that glc–oxLDL influences a broad cascade of signaling transduction pathways, which may not only result in apoptosis, but also could affect NFkB in human coronary cells. This cascade of events may influence the evolution of atherogenesis and vascular complications in diabetic patients.     

Pomegranate extract induces apoptosis in human prostate cancer cells by modulation of the IGF–IGFBP axis

The IGF axis is critical for the regulation of apoptosis in many human cancer cell lines. Recently, potent anti-tumorigenic effects of pomegranate juice and extracts have been reported. Consequently, pomegranate has potential not only as a treatment but also as a preventative measure against certain types of cancer, including prostate. In this study, we investigated the relationship between pomegranate-induced apoptosis in human prostate cancer cells and the IGF/IGFBP system. Treatment of LAPC4 prostate cancer cells with 10 μg/ml POMx, a highly potent pomegranate extract prepared from skin and arils minus seeds and standardized to ellagitannin content (37% punicalagins by HPLC), resulted in inhibition of cell proliferation and induction of apoptosis. Interestingly, co-treatment with POMx and IGFBP-3 revealed synergistic stimulation of apoptosis and additive inhibition of cell growth. Western blot analysis revealed that treatment with POMx or POMx/IGFBP-3 combination resulted in increased JNK phosphorylation, and decreased Akt and mTOR activation, consistent with a growth inhibitory, pro-apoptotic function. We also investigated the relationship between IGF-1 and pomegranate-induced apoptosis in 22RV1 prostate cancer cells. Co-treatment with 100 ng/ml IGF-1 completely blocked apoptosis induction by POMx. In contrast, IGF-I failed to inhibit POMx-induced apoptosis in R^? cells, suggesting the importance of IGF-IR. POMx-treatment decreased Igf1 mRNA expression in a dose-dependent manner indicating that its actions also involve tumor-specific suppression of IGF-1. These studies revealed novel interactions between the IGF system and pomegranate-induced apoptosis.     

An engineered cell sheet composed of human islets and human fibroblast,bone marrow–derived mesenchymal stem cells,or adipose–derived mesenchymal stem cells: An in vitro comparison study

Background: We previously reported the utility of engineered cell sheets composed of human islets and supporting cells in vitro and in vivo. It is unclear which type of supporting cell is most suitable for constructing cell sheets with human islets. The present study aimed to compare human fibroblasts, bone marrow–derived mesenchymal stem cells (BM–MSCs), and adipose–derived mesenchymal stem cells (ADSCs) as a supporting source for cell sheets.

Methods: Engineered cell sheets were fabricated with human islets using human fibroblasts, BM–MSCs, or ADSCs as supporting cells. The islet viability, recovery rate, glucose–stimulated insulin release (determined by the stimulation index), and cytokine secretion (TGF–β1, IL–6, and VEGF) of groups—including an islet–alone group as a control-were compared.

Results: All three sheet groups consistently exhibited higher viability, recovery rate, and stimulation index values than the islet-alone group. The ADSC group showed the highest viability and recovery rate among the three sheet groups. There were no discernible differences in the stimulation index values of the groups. The fibroblast group exhibited significantly higher TGF–β1 values in comparison to the other groups. The IL–6 level of the ADSC group was more than five times higher than that of the other groups. The ADSC group showed the VEGF level; however, it did not differ from that of the BM–MSC group to a statistically significant extent.

Conclusion: Engineered cell sheets composed of islets and supporting cells had a cytoprotective effect on islets. These results suggest that individual cell types could be a more attractive source for crafting engineered cell sheets in comparison to islets alone.     

Antineoplastic effects of octreotide on human gallbladder cancer cells in vitro

AIM:To investigate whether octreotide can inhibit thegrowth of human gallbladder cancer cells in vitro and toelucidate the antineoplastic mechanism of octreotide ingallbladder cancer.METHODS:A human gallbladder cancer cell line,GBC-SD,was cultured in vitro.The antiproliferative effects of octreotidewere examined by means of an MTT assay and a colony formingability assay.Morphological variation was investigated underscanning electron microscopy and transmission electronmicroscopy.Cell cycle analysis and apoptosis rate wasevaluated by flow cytometry (FCM) after staining bypropidium iodide.DNA fragmentation was assayed byagarose gel electrophoresis.Immunohistochemical stainingwas performed to evaluate the expressions of mutant-typep53 and bc1-2.RESULTS:The growth curve and colony forming abilityassay showed significant inhibition of octreotide to theproliferation of GBC-SD cells in culture in a time-and dose-dependent manner.After exposure to octreotide,GBC-SDcells showed typically apoptotic characteristics,includingmorphological changes of chromatin condensation,vacuolardegeneration,nucleus fragmentation and apoptotic bodyformation.In FCM profile apoptotic cells showed increasedsub-G_1 peaks in the octreotide group,significantly higherthan the control group (P=0.013).There was also anaugmentation in the cell proportion of G_0/G_1 phase(P=0.015),while the proportion of S phase and G_2/M phaseremained unchanged (P=0.057 and P=0.280,respectively).DNA agarose gel electrophoresis displayed a ladder afterexposure to 1000 nmol/L octreotide.After being treatedwith octreotide,the expressions of both mutant-type p53and bc1-2 decreased considering the percentage of positivecells (P<0.05).CONCLUSION:Octreotide has a negative action to theproliferation of GBC-SD cells,and the mechanism may berelated to cytostatic and cytotoxic effects.The reduction ofmutant-type p53 and bc1-2 expressions may be associatedwith the apoptosis induced by octreotide.     

The Rap–B-Raf signalling pathway is activated by glucose and glucagon-like peptide-1 in human islet cells

Aims/hypothesis Glucose and glucagon-like peptide-1 have been shown to activate extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase in beta cells. We examined the contributions of the small GTPases Rap and Ras and the serine–threonine kinases B-Raf and Raf-1 to the activation of these kinases in human islet cells.Methods The expression of Rap, Ras, B-Raf and Raf-1 in human islets was examined by immunohistochemistry and immunoblotting. Human islets were incubated in glucose at concentrations of 2.5 and 15 mmol/l and were stimulated with 10 nmol/l glucagon-like peptide-1. The activation of ERK and Raf kinases was examined by phosphorylation-specific antibodies and immuno-complexed kinase assays. The activation of Rap and Ras was determined by pull-down assays. Stimulation of phosphoinositide 3-kinase was detected by immuno-complexed lipid kinase assays.Results Extracellular-regulated kinase and protein kinase B (a downstream target of phosphoinositide 3-kinase) were activated in islets stimulated with glucose and glucagon-like peptide-1. In these islets, the Rap–B-Raf signalling pathway was activated preferentially compared with Ras and Raf-1, and activated Rap and B-Raf mediated ERK stimulation in kinase assays in vitro. In addition, Rap rather than Ras mediated activation of phosphoinositide 3-kinase in islets stimulated with glucose and glucagon-like peptide-1.Conclusions/interpretation In human islet cells, glucose and glucagon-like peptide-1 activate the Rap and B-Raf signalling module, which mediates ERK activation in assays in vitro. Rap also activates phosphoinositide 3-kinase, delineating central roles for Rap and B-Raf as therapeutic targets for beta cell growth in diabetes mellitus.J. Trümper and D. Ross contributed equally to this article     

Pyrimidine 5′-nucleotidase activity in normal and deficient human lymphoblastoid cells

Summary Two distinct pyrimidine 5-nucleotidases (UMPH-1 and UMPH-2) have previously been detected in human erythrocytes; UMPH-1 is deficient in a haemolytic anaemia, while UMPH-2 is unaffected. Only the erythrocyte shows pathological effects in this disorder. Here we have studied lymphoblastoid cell lines from control and UMPH-1 deficient patients to determine whether UMPH-1 can be detected in lymphoblastoid cells and whether the deficiency of UMPH-1 results in any measurable metabolic effects. Both UMPH-1 and a UMPH-2-like activity were found to be present in control lymphoblastoid cells. UMPH-1 was undetectable in the patients' cells; minor but significant changes were found in the pyrimidine nucleotide and nucleoside pools in the cells.     

Development and distribution of mast cells and neuropeptides in human fetus duodenum

AIM:To study the developmental regularities and heterogeneity of mast cells(MC)in human fetus duodenum and the distribution and developmental regularities of substance P(SP),calcitonin gene-related peptide(CGRP)- immunoreactive(IR)peptidergic nerves in fetus duodenum, as well as the relationship between MC,SP and CGRP-IR peptidergic nerves. METHODS:Duodena from 21 cases of human fetus and one term infant were stained by hematoxylin-eosin(HE), toluidine blue(TB)and immunohistochemical avidin-biotinylated peroxidase complex(ABC)method. RESULTS:Lobe-shape intestinal villi in duodenum were already developed at the twelfth week.At the 21st wk, muscular mucosa appeared gradually,and four layers were observed in the wall of duodenum.TB staining showed that the granules in the immature MC were pale violet, while the mature MC were strong violet in color by TB staining.Connective tissue MC(CTMC)appeared occasionally in submucosa and muscular layer of duodenum at the 16th wk.While the mucosa MC(MMC)appeared at the 18th wk.At the 22nd wk,both CTMC and MMC were activated,and distributed in the surrounding blood vessels and ganglions.The verge of some MC were unclear,and showed degranular phenomena.At the 14th wk,SP and CGRP-IR nerve fibers and cells appeared in the myenteric and submucous plexuses in small intestine,and the responses were turn strongly.Neurons were light to deep brown,and nerve fibers were present as varicose and liner profiles.On the corresponding site of serial sections,SP and CGRP immunohistochemical reactions were coexisted in one nerve fiber or cell.Some of MC showed SP and CGRP-IR positive staining. CONCLUSION:There are two heterogeneous kinds of MC in duodenum,MMC and CTMC.MC might play an important role in regulating blood circulation and sensation.     

CEA and AFP expression in human hepatoma cells transfected with antisense IGF-Igene

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IL-33 augments substance P–induced VEGF secretion from human mast cells and is increased in psoriatic skin

The peptide substance P (SP) has been implicated in inflammatory conditions, such as psoriasis, where mast cells and VEGF are increased. A relationship between SP and VEGF has not been well studied, nor has any interaction with the proinflammatory cytokines, especially IL-33. Here we report that SP (0.1–10 μM) induces gene expression and secretion of VEGF from human LAD2 mast cells and human umbilical core blood-derived cultured mast cells (hCBMCs). This effect is significantly increased by coadministration of IL-33 (5–100 ng/mL) in both cell types. The effect of SP on VEGF release is inhibited by treatment with the NK-1 receptor antagonist 733,060. SP rapidly increases cytosolic calcium, and so does IL-33 to a smaller extent; the addition of IL-33 augments the calcium increase. SP-induced VEGF production involves calcium-dependent PKC isoforms, as well as the ERK and JNK MAPKs. Gene expression of IL-33 and histidine decarboxylase (HDC), an indicator of mast cell presence/activation, is significantly increased in affected and unaffected (at least 15 cm away from the lesion) psoriatic skin, as compared with normal control skin. Immunohistochemistry indicates that IL-33 is associated with endothelial cells in both the unaffected and affected sites, but is stronger and also associated with immune cells in the affected site. These results imply that functional interactions among SP, IL-33, and mast cells leading to VEGF release contribute to inflammatory conditions, such as the psoriasis, a nonallergic hyperproliferative skin inflammatory disorder with a neurogenic component.