Abstract: PURPOSE: VEGFR-2 plays a crucial role in mediating angiogenic endothelial cell responses via the VEGF pathway and angiogenesis inhibitors targeting VEGFR-2 are in clinical use. As angiogenesis is a host-driven process, functional heritable variation in KDR, the gene encoding VEGFR-2, may affect VEGFR-2 function, and ultimately, the extent of tumor angiogenesis. EXPERIMENTAL DESIGN: We resequenced KDR using 24 DNAs each from healthy Caucasian, African American and Asian groups. Non-synonymous genetic variants were assessed for function using phosphorylation assays. Luciferase reporter gene assays were used to examine effects of variants on gene expression. KDR mRNA and protein expression, and microvessel density (MVD) were measured in non-small cell lung cancer (NSCLC) tumor samples and matching patient DNA samples were genotyped to test for associations with variants of interest.RESULTS: KDR resequencing led to the discovery of 120 genetic variants, of which 25 had not been previously reported. Q472H had increased VEGFR-2 protein phosphorylation and associated with increased MVD in NSCLC tumor samples. -2854C and -2455A increased luciferase expression and associated with higher KDR mRNA levels in NSCLC samples. -271A reduced luciferase expression and associated with lower VEGFR-2 levels in NSCLC samples. -906C and 23408G, associated with higher KDR mRNA levels in NSCLC samples. CONCLUSIONS: This study has defined KDR genetic variation in three populations and identified common variants that impact on tumoral KDR expression and vascularization. These findings may have important implications for understanding the molecular basis of genetic associations between KDR variation and clinical phenotypes related to VEGFR-2 function.
Abstract: To assess whether polymorphisms in NOD2 and ATG16L1 affect cytokine responses and mycobacterium avium subspecies paratuberculosis (MAP) survival in monocytes from Crohn's disease (CD) patients.
Abstract: Interindividual variability in the response to drug therapy is due, in part, to genetic mechanisms which influence the expression of genes involved with drug metabolism and transport. Genetic elements and processes such as DNA methylation, histone deacetylation, transcription factors, DNA sequence variants, and microRNAs (miRNAs) can impact at either the transcriptional or translational levels to modulate gene expression. Identification of such genetic regulators has greatly advanced in the last decade. Genome-wide analyses, using different types of approaches and methodologies, have uncovered many potential regulators of the expression of drug metabolizing enzymes and transporters. However, confirming the function of these putative regulators is necessary and requires further work in the laboratory, using techniques which are still evolving. It also still remains to be seen whether these findings have clinical implications for drug therapy but the realization of personalized medicine is a possible consequence of this research.
Abstract: To identify genes that may be relevant to the molecular action of antidepressants, we investigated transcriptional changes induced by the selective serotonin reuptake inhibitor paroxetine in a serotonergic cell line. We examined gene expression changes after acute treatment with paroxetine and sought to validate microarray results by quantitative PCR (qPCR). Concordant transcriptional changes were confirmed for 14 genes by qPCR and five of these, including the adrenomedullin gene (Adm), either approached or reached statistical significance. Reporter gene assays showed that a SNP (rs11042725) in the upstream flanking region of ADM significantly altered expression. Association analysis demonstrated rs11042725 to be significantly associated with response to paroxetine (odds ratio=0.075, P<0.001) but not with response to either fluoxetine or citalopram. Our results suggest that ADM is involved with the therapeutic efficacy of paroxetine, which may have pharmacogenetic utility.
Abstract: Hes6 is a neurogenic gene which is down-regulated in the hippocampi of rats chronically treated with the antidepressant paroxetine. To assess whether variability in HES6 associates with mood disorder diagnosis or antidepressant response, this gene was sequenced in 24 unrelated New Zealand Caucasians. A total of 12 polymorphisms were identified, six of which were in the promoter region of the gene. Haplotypes encompassing the promoter SNPs were studied by cloning the region upstream of the transcription start site, and examining basal transcription rates in luciferase reporter gene assays. SNPs located at positions -1099, -831, -424 and -267 were shown to significantly alter expression of the reporter gene. These four variants were tested for association with mood disorder diagnosis or antidepressant response in a family study of depression, but no significant associations were observed. However, given the importance of this gene in neural function and development, the promoter variants described here may be of wider relevance.
Abstract: AbstractAntidepressant drugs can exert significant effects on the mood of a patient suffering major depression and other disorders. These drugs generally have pharmacological actions on the uptake or metabolism of the neurotransmitters serotonin, noradrenaline and, to a lesser extent, dopamine. However, there are many aspects of antidepressant action we do not understand. We have applied proteomic analysis in a rat hippocampal model in an attempt to identify relevant molecules that operate in pathways functionally relevant to antidepressant action. Rats were administered either 5 mg/kg daily of the antidepressant paroxetine or vehicle for 12 days, then hippocampal protein was recovered and resolved by 2-D gel electrophoresis. After antidepressant exposure, we observed increased expression or modification of cytochrome c oxidase, subunit Va, cyclin-dependent kinase inhibitor 2A interacting protein, dynein, axonemal, heavy polypeptide 3 and RHO GDP-dissociation inhibitor alpha. Decreased expression or modification was observed for complexin 1 (CPLX1), alpha-synuclein, parvalbumin, ribosomal protein large P2, prohibitin, nerve growth factor, beta subunit (NGFB), peroxiredoxin 6 (PRDX6), 1-acylglycerol-3-phosphate O-acyltransferase 2_predicted, cystatin B (CYTB) and lysosomal membrane glycoprotein 1. CPLX1, the most strongly regulated protein observed, mediates the fusion of cellular transport vesicles with their target membranes and has been implicated in the pathophysiology of mood disorders, as well as antidepressant action. CPLX1 and the other proteins identified may represent links into molecular processes of importance to mood dysregulation and control, and their respective genes may represent novel candidates for studies of antidepressant pharmacogenetics.
Abstract: Antidepressant drugs can have significant effects on the mood of a patient suffering from major depression or other disorders. The pharmacological actions of these drugs generally affect the uptake or metabolism of the neurotransmitters serotonin, noradrenalin, and, to a lesser extent, dopamine. However, many aspects of antidepressant action are not understood. We conducted a proteomic analysis in a neuronal cell culture model in an attempt to identify molecules important to the operation of pathways functionally relevant to antidepressant action. The model involved generating cultures containing mixed neural and glial cells by controlled differentiation of mouse embryonic stem cells, followed by exposure to 1 microM paroxetine for 14 days. After antidepressant exposure, we observed increased expression or modification of sepiapterin reductase (SPR), heat shock protein 9A, RAS and EF-hand domain containing, and protein disulfide isomerase associated 3 and decreased expression or modification of creatine kinase, actin, prohibitin, a T-cell receptor alpha chain, defensin-related cryptdin 5, and the intermediate filament proteins glial fibrillary acidic protein and vimentin. SPR, the most strongly up-regulated protein observed, controls production of tetrahydrobiopterin, an essential cofactor for the synthesis of many neurotransmitters including serotonin, making it a plausible and intriguing candidate protein for involvement in mood control and antidepressant drug action. SPR and the other proteins identified may represent links to molecular processes of importance to mood dysregulation and control, and their respective genes may be novel candidates for the study of antidepressant pharmacogenetics.
Abstract: The World Congress of Psychiatric Genetics (WCPG) has become an annual event since the early 1990's sponsored by the International Society of Psychiatric Genetics (ISPG). Each year the latest published and unpublished findings are aired for discussion by representatives of the majority of research programs on this topic world-wide. The 2007 congress was held in New York City and attracted over 1000 researchers. The topics emphasized included results from whole genome association studies, the significance of copy number variation and the important contributions of epigenetic events to psychiatric disorders. There were over 20 oral sessions devoted to these and other topics of interest. Young investigator recipients of travel awards served as rapporteurs to summarize sessions and these summaries follow.
Abstract: McHugh PC, Rogers GR, Glubb DM, Allington MD, Hughes M, Joyce PR, Kennedy MA. Downregulation of Ccnd1 and Hes6 in rat hippocampus after chronic exposure to the antidepressant paroxetine. The mechanism of action of antidepressant drugs is not fully understood. Application of genomic methods enables the identification of biochemical pathways that are regulated by antidepressants, and this may provide novel clues to the molecular and cellular actions of these drugs. The present study examined gene expression profiles in the hippocampus of rats exposed to chronic antidepressant treatment. Animals were treated for 12 days with the selective serotonin reuptake inhibitor paroxetine; then, hippocampal ribonucleic acid was recovered, and changes in gene expression were assessed by microarray analysis. A total of 160 genes that showed differential expression after paroxetine exposure were identified. Using functional relevance and observed fold change as selection criteria, the expression changes in a subset of these genes were confirmed by quantitative polymerase chain reaction. Of this subset, only two genes, cyclin D1 (Ccnd1) and hairy and enhancer of split 6 (Hes6), showed robust and consistent changes in expression. Both genes were downregulated by paroxetine, and both have been previously implicated in neurogenesis. Further investigation of these two genes may provide new insight into the mechanism of action of antidepressants.
Abstract: Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.
Abstract: Interferon-gamma stimulation of human macrophages causes the synthesis and release of neopterin and its reduced form 7,8-dihydroneopterin (7,8-NP). The purpose of this cellular response is undetermined but in vitro experiments suggests 7,8-NP is an antioxidant. We have found 7,8-NP can protect monocyte-like U937 cells from oxidative damage. 7,8-NP inhibited ferrous ion and hypochlorite mediated loss of cell viability. Fe++ mediated lipid peroxidation was effectively inhibited by 7,8-NP, however, no correlation was found between peroxide concentration and cell viability. Hypochlorite was scavenged by 7,8-NP, preventing the loss of cell viability. 7,8-NP was less effective in inhibiting H2O2-mediated loss of cell viability with significant inhibition only occurring at high 7,8-NP concentrations. Analysis of cellular protein hydrolysates showed none of the oxidants caused the formation of any protein bound DOPA or dityrosine but did show 7,8-NP prevented the loss of cellular tyrosine by HOCl. Our data suggests macrophages may synthesize 7,8-NP for antioxidant protection during inflammatory events in vivo.
Abstract: Neopterin and the reduced form, 7,8-dihydroneopterin (78NP) are pteridines released from macrophages when stimulated with gamma-interferon in vivo. The role of 78NP in inflammatory response is unknown though neopterin has been used clinically as a marker of immune cell activation, due to its very fluorescent nature. Using red blood cells as a cellular model, we demonstrated that micromolar concentrations can inhibit or reduce red blood cell haemolysis induced by 2,2'-azobis(amidinopropane)dihydrochloride (AAPH), hydrogen peroxide, or hypochlorite. One hundred microM 78NP prevented HOCl haemolysis using a high HOCl concentration of 5 micromole HOCl/10(7) RBC. Fifty microM 78NP reduced the haemolysis caused by 2 mM hydrogen peroxide by 39% while the same 78NP concentration completely inhibited haemolysis induced by 2.5 mM AAPH. Lipid peroxidation levels measured as HPLC-TBARS were not affected by addition of 78NP. There was no correlation between lipid oxidation and cell haemolysis suggesting that lipid peroxidation is not essential for haemolysis. Conjugated diene measurements taken after 6 and 12 hour exposure to hydrogen peroxide support the TBARS data. Gel electrophoresis of cell membrane proteins indicated 78NP might inhibit protein damage. Using dityrosine as an indicator of protein damage, we demonstrated 200 microM 78NP reduced dityrosine formation in H(2) O(2) /Fe(++) treated red blood cell ghosts by 30%. HPLC analysis demonstrated a direct reaction between 78NP and all three oxidants. Two mM hydrogen peroxide oxidised 119 nM of 78NP per min while 1 mM AAPH only oxidised 50 nM 78NP/min suggesting that 78NP inhibition of haemolysis is not due to 78NP scavenging the primary initiating reactants. In contrast, the reaction between HOCl and 78NP was near instant. AAPH and hydrogen peroxide oxidised 78NP to 7,8-dihydroxanthopterin while hypochlorite oxidation produced neopterin. The cellular antioxidant properties of 78NP suggest it may have a role in protecting immune cells from free radical damage during inflammation.
