Profiling of Proteins Regulated by Venlafaxine during Neural Differentiation of Human Cells.

OBJECTIVE: Antidepressants are known to positively influence several factors in patients with depressive disorders, resulting in increased neurogenesis and subsequent relief of depressive disorders. To study the effects of venlafaxine during neural differentiation at the cellular level, we looked at its effect on protein expression and regulation mechanisms during neural differentiation. METHODS: After exposing NCCIT cell-derived EBs to venlafaxine during differentiation (1 day and 7 days), changes in protein expression were analyzed by 2-DE and MALDI-TOF MS analysis. Gene levels of proteins regulated by venlafaxine were analyzed by real-time RT-PCR. RESULTS: Treatment with venlafaxine decreased expression of prolyl 4-hydroxylase (P4HB), ubiquitin-conjugating enzyme E2K (HIP2) and plastin 3 (T-plastin), and up-regulated expression of growth factor beta-3 (TGF-ß3), dihydropyrimidinase-like 3 (DPYSL3), and pyruvate kinase (PKM) after differentiation for 1 and 7 days. In cells exposed to venlafaxine, the mRNA expression patterns of HIP2 and PKM, which function as negative and positive regulators of differentiation and neuronal survival, respectively, were consistent with the observed changes in protein expression. CONCLUSION: Our findings may contribute to improve understanding of molecular mechanism of venlafaxine.

Proteomic analysis reveals KRIT1 as a modulator for the antioxidant effects of valproic acid in human bone-marrow mesenchymal stromal cells.

Valproic acid (VPA) protects human bone marrow-mesenchymal stromal cells (hBM-MSCs) against oxidative stress and improves their migratory ability through increasing the secretion of trophic factors. This suggests that VPA may be an excellent candidate for improving stem cell function. However, the molecular mechanisms of VPA in BM-MSCs are not known. In this study, we used a proteomic approach to investigate VPA-associated targets under oxidative stress conditions. Krev/Rap1 interaction Trapped-1 (KRIT1), a modulator for the homeostasis of intracellular reactive oxygen species (ROS), was identified as a target protein by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analyses. The up-regulation of KRIT1 and its target proteins (SOD2 and FoxO1) with VPA treatment of hBM-MSCs was revealed by qPCR and immunoblot analysis. Damage from oxidative stress was reduced in VPA-pretreated BM-MSCs, which was also confirmed by qPCR and immunoblot analysis. In addition, increased in intracellular ROS by H2O2 were also reduced by VPA pretreatment in BM-MSCs. This suggests that VPA reduces intracellular ROS level by the modulation of KRIT1 and its correlated proteins, FoxO1, SOD2, and cyclin D1. Thus, this study is the first to provide evidence that VPA modulates KRIT1 and intracellular ROS in BM-MSCs.

Fluoxetine-induced regulation of heat shock protein 90 and 14-3-3ε in human embryonic carcinoma cells.

Fluoxetine, a serotonin-selective reuptake inhibitor, exerts antidepressant and antianxiety effects on major depressive and anxiety disorders. Previous studies suggest that treatment with fluoxetine influences the expression of various proteins that are involved in proliferation, differentiation, and apoptosis in the neuronal cells of the brain. However, many aspects of the molecular pathways that modulate antidepressant action are not well understood. Here, with the aim of identifying proteins involved in antidepressant action, we examined the protein expression profile of human embryonic carcinoma (NCCIT) cells in response to fluoxetine treatment using proteomic techniques such as two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). We found several upregulated and downregulated proteins in fluoxetine-treated NCCIT cells, and then biochemically confirmed the increased expression of heat shock protein 90 and 14-3-3ε, which play an essential role in many cellular mechanisms including cell cycle control and other signaling pathways. Our data suggest that the regulated expression of heat shock protein 90, 14-3-3ε, and other identified proteins may be associated with the therapeutic action of fluoxetine.

DRD3 gene rs6280 polymorphism may be associated with alcohol dependence overall and with Lesch type I alcohol dependence in Koreans.

BACKGROUND: Several polymorphisms of the dopamine D3 receptor (DRD3) gene are reported to be involved in the susceptibility to alcoholism. Although the DRD3 rs6280 (Ser9Gly) polymorphism plays an important role in various psychiatric disorders, findings regarding the association between this single-nucleotide polymorphism (SNP) and alcohol dependence (AD) have been inconsistent. Therefore, the present study investigated the association between the DRD3 gene rs6280 polymorphism with AD and Lesch type I AD in Korean subjects. METHODS: The DRD3 rs6280 SNP was genotyped in a case-control sample comprising 245 AD patients and 130 healthy controls (HCs). Alcohol Use Disorders Identification Test (AUDIT) scores were also compared relative to genotype in all of the participants. RESULTS: This SNP was significantly associated with both AD overall (χ(2) = 10.09 and p = 0.001, and χ(2) = 10.60 and p = 0.005, for the recessive and additive models, respectively) and with Lesch type I AD (χ(2) = 11.70 and p = 0.001, and χ(2) = 11.70 and p = 0.003, for the recessive and additive models, respectively). The allele frequency differed significantly (χ(2) = 8.45, p = 0.004) between Lesch type I AD and HC subjects. The AUDIT total (F = 6.56, p = 0.011), hazardous alcohol use (F = 7.12, p = 0.008), dependence symptoms (F = 5.10, p = 0.025), and harmful alcohol use (F = 4.83, p = 0.029) scores were significantly higher in those who did not possess the S allele (genotype GG) than in those who did (genotypes SS ± SG). CONCLUSIONS: The findings of this study suggest that the DRD3 rs6280 polymorphism is associated with the development of both AD overall and Lesch type I AD in Koreans.

The association of DRD2 -141C and ANKK1 TaqIA polymorphisms with alcohol dependence in Korean population classified by the Lesch typology.

AIMS: Dopamine receptors are associated with reward and dependence towards alcohol. The polymorphisms of dopamine D2 receptor (DRD2) genes have been reported to be involved in susceptibility to alcoholism. Therefore, we investigated the association of three single-nucleotide polymorphisms (SNPs) in DRD2 and ankyrin repeat and kinase domain containing one (ANKK1) genes with alcohol dependence in Korean subjects, who were classified by the criteria of the Lesch typology. METHODS: The DRD2 -141C (Insertion (Ins)/Deletion (Del)), exon8 (A/G) and the ANKK1 TaqIA (A1/A2) polymorphisms were genotyped in a case-control sample consisting of 245 alcohol-dependent (AD) patients and 110 healthy controls. AD patients were classified into four subtypes by the Lesch typology. The majority of them (77.1%) were Lesch type 1. Differences in genotype and allele frequencies were examined between the AD patients and the controls. Also those analyses were done between the Lesch type 1 group and the controls. RESULTS: There were significant differences in the genotype and allele frequencies of -141C Ins/Del and TaqIA A1/A2 between the AD patients and the controls. However, there were no significant differences in genotype or allele frequencies of exon8 A/G between the AD patients and the controls. The -141C Ins/Ins and TaqIA A1+ variants were associated with Lesch type 1 AD patients. When analysing haplotypes of three SNPs, the odds ratio of -141C Ins-A-A1 was 2.286, while the odds ratio of -141C Del-G-A2 was 0.323. CONCLUSION: The present study showed a significant difference in DRD2 -141C and ANKK1 TaqIA polymorphisms between the AD patients and the controls. Our findings suggest that -141C Ins and TaqIA A1 alleles can be a predisposing factor for alcohol dependence in the Korean population.

Proteomic analysis of the copper ion-induced stress response in a human embryonic carcinoma cell line.

Excessive exposure to copper, a redox-active metal, generates free radicals, which can cause cellular damage. In this study, we aim to identify the proteins that are up- or downregulated by copper exposure in human embryonic carcinoma (NCCIT) cells and to understand the mechanisms that play a role in the copper-induced stress response. After exposure to copper ions, the cells showed upregulated levels of 78 kDa glucose-regulated protein, fibrillin 1, CWC22 spliceosome-associated protein (KIAA1604), heat shock protein (HSP) 60, and HSP70, while the tumor necrosis factor receptor-associated factor 6, vimentin, 14-3-3 protein zeta, and RAC-beta (AKT2) serine/threonine protein kinase were downregulated. The GeneGo Process Networks of the proteins upregulated by copper ions were analyzed, and the 3 highest-scoring networks from the proteins upregulated by copper ions are presented here. In particular, the increased level of HSP70 in response to copper ions occurred in a dose-dependent manner, indicating that HSP70 could be a potential biomarker for copper toxicity in mammalian cells.

Comparative proteome analysis of Bacillus anthracis with pXO1 plasmid content.

Bacillus anthracis the causative agent of anthrax, is an important pathogen among the Bacillus cereus group of species because of its physiological characteristics and its importance as a biological warfare agent. Tripartite anthrax toxin proteins and a poly-D-glutamic acid capsule are produced by B. anthracis vegetative cells during mammalian hosts infection and when cultured in conditions that are thought to mimic the host environment. To identify the factors regulating virulence in B. anthracis the whole cell proteins were extracted from two B. anthracis strains and separated by narrow range immobilized pH gradient (IPG) strips (pH 4-7), followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins that were differentially expressed were identified by the peptide fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 23 proteins were identified as being either upregulated or downregulated in the presence or absence of the virulence plasmid pXO1. Two plasmid encoded proteins and 12 cellular proteins were identified and documented as potential virulence factors.