👤 Paula K Zhu

Hepatocellular carcinoma (HCC) is believed to arise from tumor-initiating cells (T-ICs), although little is known about their stem cell-like properties. We quantified levels of p28(GANK) (Gankyrin), OV6, and Oct4 in 130 human HCC samples using immunohistochemistry. Magnetic-activated cell sorting was used to isolate OV6+ HCC cells. T-IC properties were evaluated by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and spheroid formation. We used a coimmunoprecipitation assay to study interactions among p28(GANK), Oct4, and WWP2. Tumorigenicity and pulmonary metastasis were examined in nonobese diabetic and severe combined immunodeficient mice. In HCC samples, high levels of p28(GANK) correlated with expansion of OV6+ tumor cells; the combination of high levels of p28(GANK) and OV6 was associated with progression of HCC. p28(GANK) was predominantly expressed in liver T-ICs, isolated by magnetic sorting, and undifferentiated primary HCC spheroids. Increased levels of p28(GANK) in T-ICs increased their percentages in HCC samples, expression of stem cell genes, self-renewal potential, chemoresistance in vitro, and tumorigenicity and ability to develop into pulmonary metastases in mice. Conversely, knockdown of p28(GANK) reduced their T-IC properties. p28(GANK) likely activates liver T-ICs by impeding ubiquitination and degradation of the transcription factor Oct4 by WWP2. In support of this concept, levels of p28(GANK) correlated with those of Oct4 in HCC samples. p28(GANK) activates and maintains liver T-ICs in HCCs by preventing degradation of Oct4. Inhibitors of p28(GANK) might therefore be developed to inactivate T-ICs and slow tumor progression. Show less

Functional studies suggest that the APOA5 -1131T/C polymorphism plays an important role in triglyceride (TG) metabolism, which is an event contributing to the pathogenesis of coronary artery disease (CAD). However, genetic evidence of its effect on CAD is inconsistent. To assess this correlation, we performed a meta-analysis of published data. A comprehensive meta-analysis was performed on nine published studies, with a total sample of 2049 subjects and 2373 controls using a fixed effect model. Under the fixed effect model, the risk of the disease was significantly higher in subjects with CC genotype in comparison with both TT (OR: 1.99; 95% CI: 1.64-2.41) and TC (OR: 1.48; 95% CI: 1.22-1.80) subjects. Compared with TT homozygotes, there was 43% increase in the incidence of CAD (OR: 1.43; 95% CI: 1.26-1.61) of C carriers (CC+TC). There was no heterogeneity for these effect estimates. Our findings support the view that -1131T/C polymorphism of the APOA5 gene is associated with CAD and the C allele might be a genetic risk factor that increases susceptibility to CAD. Show less

Neuronal nitric oxide synthase (nNOS) is mainly expressed in neurons, to some extent in astrocytes and neuronal stem cells. The alternative splicing of nNOS mRNA generates 5 isoforms of nNOS, including nNOS-α, nNOS-β, nNOS-µ, nNOS-γ and nNOS-2. Monomer of nNOS is inactive, and dimer is the active form. Dimerization requires tetrahydrobiopterin (BH4), heme and L-arginine binding. Regulation of nNOS expression relies largely on cAMP response element-binding protein (CREB) activity, and nNOS activity is regulated by heat shock protein 90 (HSP90)/HSP70, calmodulin (CaM), phosphorylation and dephosphorylation at Ser847 and Ser1412, and the protein inhibitor of nNOS (PIN). There are primarily 9 nNOS-interacting proteins, including post-synaptic density protein 95 (PSD95), clathrin assembly lymphoid leukemia (CALM), calcium/calmodulin-dependent protein kinase II alpha (CAMKIIA), Disks large homolog 4 (DLG4), DLG2, 6-phosphofructokinase, muscle type (PFK-M), carboxy-terminal PDZ ligand of nNOS (CAPON) protein, syntrophin and dynein light chain (LC). Among them, PSD95, CAPON and PFK-M are important nNOS adapter proteins in neurons. The interaction of PSD95 with nNOS controls synapse formation and is implicated in N-methyl-D-aspartic acid-induced neuronal death. nNOS-derived NO is implicated in synapse loss-mediated early cognitive/motor deficits in several neuropathological states, and negatively regulates neurogenesis under physiological and pathological conditions. Show less

Multiple osteochondromas (MO), an inherited autosomal dominant disorder, is characterized by the presence of multiple exostoses on the long bones. MO is caused by mutations in the EXT1 or EXT2 genes which encode glycosyltransferases implicated in heparin sulfate biosynthesis. In this study, efforts were made to identify the underlying disease-causing mutations in patients from two MO families in China. Two novel EXT1 gene mutations were identified and no mutation was found in EXT2 gene. The mutation c.497T > A in exon 1 of the EXT1 gene was cosegregated with the disease phenotype in family 1 and formed a stop codon at amino acid site 166. The fetus of the proband was diagnosed negative. In family 2, the mutation c.1430-1431delCC in exon 6 of the EXT1 gene would cause frameshift and introduce a premature stop codon after the reading frame being open for 42 amino acids. The fetus of this family inherited this mutation from the father. Mutation analysis of two MO families in this study demonstrates its further application in MO genetic counseling and prenatal diagnosis. Show less

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a "second hit," that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome. Show less

Liver X receptor α (LXRα) and sterol regulatory element binding protein-1c (SREBP-1c) were studied in rats with non-alcoholic steatohepatitis (NASH) induced by a high-fat diet. Forty 5-week-old rats were fed either a high-fat diet (n = 30) or a normal diet (n = 10) for 9, 13 or 17 weeks. The mRNA and protein levels for LXRα and SREBP-1c were measured at each time point, as was fatty acid synthase (FAS) activity and the serum levels of free fatty acid (FFA) and triglyceride (TG). The mRNA and protein levels for LXRα and SREBP-1c, FAS activity and serum levels of FFA and TG all significantly increased from week 9 in the high-fat diet rats versus controls. In conclusion, a high-fat diet upregulates LXRα which, in turn, upregulates SREBP-1c, increasing the activity of FAS and FFA and accumulation of TG in hepatocytes. Thus, LXRα and SREBP-1c contribute to the development of NASH. Show less

MiR-140 is a microRNA specially involved in chondrogenesis and osteoarthritis pathogenesis. However, its transcriptional regulation and target genes in cartilage development are not fully understood. Here we detected that miR-140 was uniquely expressed in chondrocyte and suppressed by Wnt/β-catenin signalling. The miR-140 primary transcript was an intron-retained RNA co-expressed with Wwp2-C isoform, which was directly induced by Sox9 through binding to the intron 10 of Wwp2 gene. Knockdown of miR-140 in limb bud micromass cultures resulted in arrest of chondrogenic proliferation. Sp1, the activator of the cell cycle regulator p15(INK4b), was identified as a target of miR-140 in maintaining the chondrocyte proliferation. Collectively, our findings expand our understanding of the transcriptional regulation and the chondrogenic role of miR-140 in chondrogenesis. Show less

Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gut-derived incretin hormones that regulate blood glucose levels. In addition to their widely accepted insulinotropic role, there is evidence that GLP-1 modulates feeding behavior and GIP regulates lipid metabolism, thereby promoting postprandial fat deposition. In this study, we investigated whether naturally occurring polymorphisms in the GLP-1 receptor (GLP-1R) and the GIP receptor (GIP-R) affect the pharmacological properties of these proteins. After transient expression of the receptors in human embryonic kidney 293 cells, basal and ligand-induced cAMP production were assessed by use of luciferase reporter gene assays. Our data reveal that the wild-type GIP-R displays a considerable degree of ligand-independent activity. In comparison, the GIP-R variants C46S, G198C, R316L, and E354Q show a marked decrease in basal signaling that may, at least in part, be explained by reduced cell surface expression. When stimulated with GIP, the C46S and R316L mutants display significantly reduced potency (>1000 and 25- fold, respectively) compared with wild type. Complementary competition binding assays further demonstrate that the C46S variant fails to bind radio-iodinated GIP, whereas all other GIP-R mutants maintain normal ligand affinity. In contrast to the GIP-R, the wild-type GLP-1R lacks constitutive activity. Furthermore, none of the 10 GLP-1R missense mutations showed an alteration in pharmacological properties versus wild type. The extent to which abnormalities in GIP-R function may lead to physiological changes or affect drug sensitivity in selected populations (e.g., obese, diabetic individuals) remains to be further investigated. Show less

It has been reported that N-methyl-D-aspartate receptor (NMDAR)-triggered neurotoxicity is related to excessive Ca(2+) loading and an increase in nitric oxide (NO) concentration. However, the molecular mechanisms that underlie these events are not completely understood. NMDARs and neuronal NO synthase each binds to the scaffolding protein postsynaptic density (PSD)-93 through its PDZ domains. In this study, we determined whether PSD-93 plays a critical role in NMDAR/Ca(2+)/NO-mediated neurotoxicity. We found that the targeted disruption of the PSD-93 gene attenuated the neurotoxicity triggered by NMDAR activation, but not by non-NMDAR activation, in cultured mouse cortical neurons. PSD-93 deficiency reduced the amount of NMDAR subunits NR2A and NR2B in synaptosomal fractions from the cortical neurons and significantly prevented NMDA-stimulated increases in cyclic guanosine 3',5'-monophosphate and Ca(2+) loading in the cortical neurons. These findings indicate that PSD-93 deficiency could block NMDAR-triggered neurotoxicity by disrupting the NMDAR-Ca(2+)-NO signaling pathway and reducing expression of synaptic NR2A and NR2B. Since NMDARs, Ca(2+), and NO play a critical role during the development of brain trauma, seizures, and ischemia, the present work suggests that PSD-93 might contribute to molecular mechanisms of neuronal damage in these brain disorders. Show less

The functions of distinct isoforms of solute carrier family 27 transporters (SLC27A1-6), acetyl-CoA carboxylase (ACACA, ACACB), stearoyl-CoA desaturase (SCD1-4), fatty acid desaturase (FADS1-3), LPIN (LPIN1-3), insulin-induced gene (INSIG1, 2), and peroxisome proliferator-activated receptor gamma coactivator1 (PPARGC1A, B) were studied in the mouse mammary gland from pregnancy to lactation. The relative mRNA abundance and percent change in real-time PCR were determined. mRNA expression of SLC27A3 and SLC27A4 was 37- and 1.4-fold more upregulated at 12 days of lactation, respectively (P < 0.01). Transcripts of SCD isoforms were the most abundant, accounting for 59% of all genes measured, and PPARGC1 isoforms were the least (0.06% of all genes measured). The mRNA abundance from ACC, FADS and LPIN accounted for 29, 9 and 2.6%, respectively. INSIG1 mRNA expression was 32-fold more upregulated (P < 0.05), while PPARGC1B was 0.18-fold downregulated at 18 days of lactation (P < 0.01). We concluded that mRNA abundance and expression of these isoforms are affected by the stage of lactation. Show less

The immunotoxicity of tributyltin (TBT) on marine gastropods has been comparatively little studied although risks to wildlife associated with this compound are well known. In this study, a 30-day trial was conducted to evaluate the immunotoxic effects on abalone (Haliotis diversicolor supertexta) by exposing a range of doses of TBT (0, 2, 10, and 50 ng/L). Innate immune parameters, including phagocytic ability (PA), lysozyme activity, phenoloxidase (PO) level and superoxide dismutase (SOD) activity were monitored at intervals of 5, 15 and 30 days. Haemolymph protein expression profile was also examined at the end of the experiment. The results showed that PA value, lysozyme activity and PO level significantly decreased compared with the controls (P < 0.05), which indicated that TBT exposure markedly suppressed non-specific immune competence. Exposure to TBT also caused variation in protein expression patterns of haemolymph. Among the protein spots of differential expressions, seven proteins from the haemolymph of TBT-treated abalone were successfully identified by MALDI-TOF-MS analysis. Three protein spots increased and were identified as carrier-like peptide, peroxidase 21 precursor and creatine phosphokinase. These proteins are believed to up-regulate in expression as a response to detoxification and antioxidative stress mechanisms. The other four protein spots that down-regulated in TBT-treated groups were identified as aromatase-like protein, protein kinase C, ceruloplasmin and microtubule-actin crosslinking factor 1, and these proteins play an important role in endocrine regulation and immune defense. Taken together, the results demonstrate that TBT impair abalone immunological ability and is a potential immune disruptor. Show less

The Forkhead transcription factors FoxO1, FoxO3a, and FoxO4 play a prominent role in regulating cell survival and cell cycle. Whereas FOXO1 was shown to mediate insulin sensitivity and adipocyte differentiation, the role of the transcription factor FoxO4 in metabolism remains ill defined. To uncover the effects of FoxO4, we generated a cellular model of stable FoxO4 overexpression and subjected it to microarray-based gene expression profiling. While pathway analysis revealed a disruption of cholesterol biosynthesis gene expression, biochemical studies revealed an inhibition of cholesterol biosynthesis, which was coupled with decreased mRNA levels of lanosterol 14alpha demethylase (CYP51). FoxO4-mediated repression of CYP51 led to the accumulation of 24,25 dihydrolano-sterol (DHL), which independently and unlike lanosterol inhibited cholesterol biosynthesis. Furthermore, FoxO4-overexpressing cells accumulated lipid droplets and triacylglycerols and had an increase in basal glucose uptake. Recapitulation of these effects was obtained following treatment with CYP51 inhibitors, which also induce DHL buildup. Moreover, DHL but not lanosterol strongly stimulated liver X receptor alpha (LXRalpha) activity, suggesting that DHL and LXRalpha mediate the downstream effects initiated by FoxO4. Together, these studies suggest that FoxO4 acts on CYP51 to regulate the late steps of cholesterol biosynthesis. Show less

Cell polarity proteins regulate tight junction formation and directional migration in epithelial cells. To date, the mechanism by which these polarity proteins assemble at the leading edge of migrating epithelial cells remains unclear. We report that occludin, a transmembrane protein, is localized at the leading edge of migrating cells and regulates directional cell migration. During migration, occludin knockdown disrupted accumulation of aPKC-Par3 and PATJ at the leading edge, and led to a disorganized microtubule network and defective reorientation of the microtubule organization center (MTOC). Phosphorylation of occludin at tyrosine 473 residue allowed recruitment of p85 alpha to the leading edge via association with its C-terminal SH2 domain. Loss of occludin attenuated activation of PI3K, leading to disorganization of the actin cytoskeleton and reduced cell protrusions. Our data indicate that occludin is required for the leading-edge localization of polarity proteins aPKC-Par3 and PATJ and promotes cell protrusion by regulating membrane-localized activation of PI3K. Show less

To detect gene mutations associated with hypertrophic cardiomyopathy (HCM) in Chinese patients and possible correlations between genotype and phenotype. Twenty-one unrelated patients with hypertrophic cardiomyopathy were studied. The clinical data including symptoms, physical examination, echocardiography and electrocardiography were collected. The full ecoding exons of cardiac myosin-binding protein C gene (cMYBPC3) were amplified with PCR and the products were sequenced. Two mutations were identified in probands from two families. One mutation was frame shift mutation Pro1208fs in the exon 32 of the cMYBPC3 gene. Pro1208fs mutation was identified in a 59 years old female patient with familial hypertrophic cardiomyopathy. Symptom onset was late and a favorable clinical course was evidenced in this patient. Another mutation was missence mutation Gly507Arg in the exon 17 of the MYBPC3 gene identified in a 24 years old male patient. Diffuse thickness of left ventricular wall, impaired diastolic function and enlarged left atria were evidenced in echocardiography. No mutation was identified in the 80 control healthy individuals. cMYBPC3 might be the disease-causing genes in Chinese patients with hypertrophic cardiomyopathy. Show less

To screen the MYBPC3 gene mutations in Han Chinese patients with hypertrophic cardiomyopathy (HCM). Sixty-six patients with HCM were enrolled for the study. The exons in the functional regions of MYBPC3 were amplified with PCR and the products were sequenced. Four novel mutations and four common polymorphisms were identified in this patient cohort. A Lys301fs mutation in exon10 was evidenced in a H30, and when he was 47 years old, he had the chest tightness, shortness of breath with septal hypertrophy of 18.7mm; a Asp463stop mutation in exon17 was detected in a H48, he was 24 years old 24-year-old when a medical examination showed ventricular septal hypertrophy of 15.4 mm; both Gly523Arg mutation in exon18 and Tyr847His mutation in exon26 were found in a H53 with onset age 36 years old, feeling chest tightness after excise and his ventricular septal hypertrophy was 27 mm that time. MYBPC3 mutations occurred in 4.5% patients in this cohort. These mutations were not found in 100 non-HCM control patients. MYBPC3 mutation is presented in a small portion of Han Chinese patients with HCM. Show less

Atherosclerosis is the primary independent risk factor of cardiovascular disease, and Liver X Receptors (LXRalpha and LXRbeta) activation may play an anti-atherosclerosis effect. In this article, we summarize the current state of knowledge of roles of LXRs in physiology and homeostasis as well as the links between LXR action and atherosclerosis, and discuss the potential therapeutic effects of LXR agonists. A MEDLINE database search was performed to identify relevant articles using the keywords "liver X receptors", "LXRs", and "atherosclerosis". Additional papers were identified by a manual research of the references from the key articles. Both LXR isoforms promote reverse cholesterol transport (RCT) and have anti-inflammatory activity. LXRalpha is the predominant receptor in the liver regulating triglyceride synthesis. The antiatherosclerotic ability of LXRs makes them attractive targets for drugs for the treatment of cardiovascular disease. However, LXR activation induces lipogenesis and hypertriglyceridemia. The first-generation synthetic ligands of LXR increase hepatic lipogenesis and plasma triglyceride levels. New LXR ligands need to be designed without undesirable side effects. LXR beta-selective agonists and LXR modulators, which act as agonists in macrophages and induce cholesterol efflux while as antagonists of lipogenesis in the liver, are two critical and attractive approaches to treat atherosclerosis and cardiovascular diseases. Show less

The liver X receptors (LXRs) sense oxysterols and regulate genes involved in cholesterol metabolism. Synthetic agonists of LXRs are potent stimulators of fatty acid synthesis, which is mediated largely by sterol regulatory element-binding protein-1c (SREBP-1c). Paradoxically, an improved hepatic lipid profile by LXR was observed in mice fed a Western high fat (HF) diet. To explore the underlying mechanism, we administered mice normal chow or an HF diet and overexpressed LXRalpha in the liver. The HF diet with tail-vein injection of adenovirus of LXRalpha increased the expression of LXR-targeted genes involved in cholesterol reverse transport but not those involved in fatty acid synthesis. A similar effect was also observed with the use of 22R-hydroxycholesterol, an LXR ligand, in cultured hepatocytes. Consequently, SREBP-1c maturation was inhibited by the HF diet, which resulted from the induction of Insig-2a. Importantly, increased cholesterol level suppressed the expression of 2,3-oxidosqualene cyclase (OSC), which led to an increase in endogenous LXR ligand(s). Furthermore, siRNA-mediated knockdown of OSC expression enhanced LXR activity and selectively up-regulated LXR-targeted genes involved in cholesterol reverse transport. Thus, down-regulation of OSC may account for a novel mechanism underlying the LXR-mediated lipid metabolism in the liver of mice fed an HF diet. Show less

Liver X receptors (LXRs) are important transcriptional regulators of lipid homeostasis and proliferation in several cell types. However, the roles of LXRs in pancreatic beta cells have not been fully established. The aim of this study was to investigate the effects of LXRs on pancreatic beta cell proliferation. Gene expression was analysed using real-time RT-PCR. Transient transfection and reporter gene assays were used to determine the transcriptional activity of LXRs in pancreatic beta cells. Cell viability and proliferation were analysed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DNA fluorometric, BrdU labelling and [(3)H]thymidine incorporation assays. Cell cycle distribution was investigated by flow cytometry analysis. Adenovirus-based RNA interference was used to knockdown LXRalpha, LXRbeta and p27 in MIN6 cells and mouse islets. We found that both Lxralpha (also known as Nr1h3) and Lxrbeta (also known as Nr1h2) were expressed and transactivated the LXR response element in HIT-T15 and MIN6 cells. Activation of LXRs dose-dependently inhibited pancreatic beta cell viability and proliferation. This was accompanied by beta cell cycle arrest at the G1 phase. Furthermore, LXR activation increased levels of the p27 protein by inhibiting its degradation. Knockdown of p27 reversed these effects of LXR activation on growth inhibition and cell cycle arrest. Our observations indicate that LXR activation inhibits pancreatic beta cell proliferation through cell cycle arrest. A well-known regulator of pancreatic beta cell cycle progression, p27, is upregulated and mediates the effects of LXRs on growth inhibition in beta cells. These observations suggest the involvement of aberrant activation of LXR in beta cell mass inadequacy, which is an important step in the development of type 2 diabetes. Show less

Histone deacetylase inhibitor (HDACi) has been shown to demethylate the mammalian genome, which further strengthens the concept that DNA methylation and histone modifications interact in regulation of gene expression. Here, we report that an HDAC inhibitor, depsipeptide, exhibited significant demethylating activity on the promoters of several genes, including p16, SALL3, and GATA4 in human lung cancer cell lines H719 and H23, colon cancer cell line HT-29, and pancreatic cancer cell line PANC1. Although expression of DNA methyltransferase 1 (DNMT1) was not affected by depsipeptide, a decrease in binding of DNMT1 to the promoter of these genes played a dominant role in depsipeptide-induced demethylation and reactivation. Depsipeptide also suppressed expression of histone methyltransferases G9A and SUV39H1, which in turn resulted in a decrease of di- and trimethylated H3K9 around these genes' promoter. Furthermore, both loading of heterochromatin-associated protein 1 (HP1alpha and HP1beta) to methylated H3K9 and binding of DNMT1 to these genes' promoter were significantly reduced in depsipeptide-treated cells. Similar DNA demethylation was induced by another HDAC inhibitor, apicidin, but not by trichostatin A. Our data describe a novel mechanism of HDACi-mediated DNA demethylation via suppression of histone methyltransferases and reduced recruitment of HP1 and DNMT1 to the genes' promoter. Show less

Hepatocyte paraffin 1 (Hep Par 1), a murine monoclonal antibody, is widely used in surgical pathology practice to determine the hepatocellular origin of neoplasms. However, identity of the antigen for Hep Par 1 is unknown. The aim of this study was to characterize the Hep Par 1 antigen. To identify the antigen, immunoprecipitation was used to isolate the protein from human liver tissue, and a distinct protein band was detected at approximately 165 kDa. The protein band was also present in small intestinal tissue, but was not present in several other non-liver tissues nor in three human hepatocellular carcinoma cell lines, Huh-7, HepG2, and LH86. The protein was purified and analyzed by mass spectrometry. It was identified as carbamoyl phosphate synthetase 1 (CPS1). CPS1 is a rate-limiting enzyme in urea cycle and is located in mitochondria. We demonstrated that hepatoid tumors (gastric and yolk sac) were immunoreactive with both Hep Par 1 antibody and anti-CPS1 antibody, further confirming the results of mass spectrometric analysis. We found that the three human hepatocellular carcinoma cell lines do not express either CPS1 RNA or protein. We confirmed that the gene was present in these cell lines, suggesting that suppression of CPS1 expression occurs at the transcriptional level. This finding may have relevance to liver carcinogenesis, since poorly differentiated hepatocellular carcinomas exhibit poor to absent immunoreactivity to Hep Par 1. In conclusion, we have identified the antigen for Hep Par 1 antibody as a urea cycle enzyme CPS1. Our results should encourage further investigation of potential role that CPS1 expression plays in liver pathobiology and carcinogenesis. Show less

Postsynaptic density (PSD)-93, a neuronal scaffolding protein, binds to and clusters N-methyl-D-aspartate receptor (NMDAR) subunits NR2A and NR2B at cellular membranes in vitro. However, the roles of PSD-93 in synaptic NR2A and NR2B targeting in the central nervous system and NMDAR-dependent physiologic and pathologic processes are still unclear. We report here that PSD-93 deficiency significantly decreased the amount of NR2A and NR2B in the synaptosomal membrane fractions derived from spinal cord dorsal horn and forebrain cortex but did not change their levels in the total soluble fraction from either region. However, PSD-93 deficiency did not markedly change the amounts of NR2A and NR2B in either synaptosomal or total soluble fractions from cerebellum. In mice deficient in PSD-93, morphine dose-dependent curve failed to shift significantly rightward as it did in wild type (WT) mice after acute and chronic morphine challenge. Unlike WT mice, PSD-93 knockout mice also showed marked losses of NMDAR-dependent morphine analgesic tolerance and associated abnormal sensitivity in response to mechanical, noxious thermal, and formalin-induced inflammatory stimuli after repeated morphine injection. In addition, PSD-93 knockout mice displayed dramatic loss of jumping activity, a typical NMDAR-mediated morphine withdrawal abstinence behavior. These findings indicate that impaired NMDAR-dependent neuronal plasticity following repeated morphine injection in PSD-93 knockout mice is attributed to PSD-93 deletion-induced alterations of synaptic NR2A and NR2B expression in dorsal horn and forebrain cortex neurons. The selective effect of PSD-93 deletion on synaptic NMDAR expression in these two major pain-related regions might provide the better strategies for the prevention and treatment of opioid tolerance and physical dependence. Show less

Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-alpha. Additionally, the relationship between TNF-alpha resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated. To compare differences in the proteome of the TNF-alpha resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-alpha resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT. Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (delta-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression. This study demonstrates that MEK5 over-expression promotes a TNF-alpha resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression. Show less

Anthocyanins belong to a large and widespread group of water-soluble phytochemicals and exhibit potent antioxidative and anti-inflammatory properties; however, the molecular mechanisms of these biochemical actions mediated by anthocyanins remain unclear. In this study, our data show that pretreatment of THP-1 macrophages with Cyanidin-3-O-beta-glucoside (C3G) for 12 h can enhance the expression and transcriptional activities of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha). Furthermore, pretreatment of these cells with C3G for 12 h causes dose-dependent inhibition of lipopolysaccharide (LPS)-induced nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both the mRNA and protein levels together with a decrease in nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production. Consequently, addition of geranylgeranyl pyrophosphate ammonium salt (GGPP), an LXRalpha antagonist, significantly downregulates the inhibitory effect of C3G on LPS-induced iNOS and COX-2 expression in THP-1 macrophages, whereas the PPARgamma antagonist GW9662 has no effect. Further investigation revealed that LXRalpha might interfere with LPS-induced iNOS and COX-2 expression by suppressing the functional activation of nuclear factor-kappaB (NF-kappaB), not - as was previously proposed - by reducing NF-kappaB nuclear translocation. Taken together, these results indicate that LXRalpha activation has an essential role in the anti-inflammatory property of C3G. Moreover, they provide new insight into the molecular basis for the anti-inflammatory property of anthocyanins. Show less

The liver X receptors (LXRs) regulate a set of genes involved in lipid metabolism and reverse cholesterol transport. We investigated the mechanism by which shear stress regulates LXR in vascular endothelial cells (ECs). Western blot showed that the protein level of LXRalpha and its target ABCA1 in the mouse thoracic aorta was higher than that in the aortic arch. As well, the mRNA level of LXR and its target genes ABCA1, ABCG1, ApoE, and LPL in the thoracic aorta was higher. In vitro, bovine aortic ECs were subjected to a steady laminar flow (12 dyne/cm2). The expressions of LXR and the LXR-mediated transcription were increased by laminar shear stress. Laminar flow increased LXR-ligand binding and the gene expression of sterol 27-hydroxylase (CYP27), which suggests an increased level of LXR ligand in ECs. This effect was attenuated by LXRalpha and CYP27 RNAi. The decrease of LXR in the aorta of PPARgamma+/- mice and that of C57 mice fed with PPARgamma antagonist suggest the involvement of PPARgamma in the LXR induction by flow. Laminar flow increases LXR function via a PPARgamma-CYP27 dependent mechanism, which reveals an atheroprotective role for laminar flow exerting on endothelium. Show less

Transduction of developmental and environmental cues into the nucleus to induce transcription and the export of RNAs to the cytoplasm through the nuclear pore complex (NPC) play pivotal roles in regulation of gene expression. The process of bulk export of mRNAs from nucleus to cytoplasm is highly conserved across eukaryotes. Assembly of export-competent mRNA ribonucleoprotein (mRNP) is coupled with both transcription and mRNA processing. The export-competent mRNP consists of mRNAs and a dozen nucleocytoplasmic shuttling nuclear proteins, including RNA export factors (Mex67-Mtr2 heterodimer, Npl3), poly(A)-binding proteins, DEAD-box protein 5 (Dbp5), and nucleoporins (NUPs) in yeast. Mobile NUPs help docking of mRNP to the NPC nuclear basket. A partially unfolded mRNP complex appears to be pulled through the NPC by using energy from Dbp5-catalyzed ATP hydrolysis. Dbp5 probably catalyzes the release of mRNA from mRNP in the cytoplasm. In contrast to bulk export of mRNAs by a Mex67-Mtr2/Npl3-dependent pathway, a specific subset of mRNA export under stress and export of microRNAs are mediated through the karyopherin (importin beta) family of proteins in a Ran-GTPase-dependent pathway. Our knowledge of mRNA export mechanisms in flowering plants is in its infancy. Some proteins of the NUP107-160 complex, NUPs and DEAD-box proteins (DBPs), have been studied in flowering plants. Arabidopsis NUP160/SAR1 plays a critical role in mRNA export, regulation of flowering, and hormone and abiotic stress responses, whereas NUP96/ SAR3/MOS3 is required for mRNA export to modulate hormonal and biotic stress responses. DEAD-box proteins have been implicated in mRNA export and abiotic stress response of yeast and higher plants. Arabidopsis DBP CRYOPHYTE/LOS4 plays an important role in mRNA export, abiotic stress response, germination, and plant development. Further studies on various components of nuclear mRNA export in plants during nonstress and stress conditions will be necessary to understand the link between mRNA export and stress-responsive gene expression. Show less

The PIK3C3 gene has been implicated as a candidate gene for schizophrenia by functional evidence and genetic association studies. A series of previous studies have found susceptibility SNPs in promoter region. To further verify its susceptibility to schizophrenia in the Chinese population and the function of the polymorphisms, we performed a case control study in 556 unrelated schizophrenia patients and 563 normal controls as well as an in vitro functional analysis. In our association analysis of-432C-/T, we discovered obvious differences in allele frequency between patients and controls (P=0.017). A T/C haplotype constructed by -432C-/T and -86insC, which are tightly linked with each other (r(2)=1) can significantly weaken promoter's transcriptional activity by 20% (p=0.002 by t-test). Though we cannot exclude the possibility that susceptibility of -432C-/T is caused by its linkage disequilibrium with other causal variants, our results do support PIK3C3 play a significant role in the etiology of schizophrenia. Show less

Increased protein level in the cerebrospinal fluid (CSF) is a characteristic of patients with Guillain-Barré syndrome (GBS), an acute inflammatory autoimmune disorder in the peripheral nervous system (PNS). However, the molecular mechanisms underlying the disease remain poorly understood and so far no reliable disease-related markers are available. By comparing the CSF proteome of GBS patients with control subjects suffering from other neurological disorders, it may be possible to identify proteins that involve in the disease process and thus to study the pathogenesis of GBS. We used two-dimensional difference gel electrophoresis (2D DIGE) technique, in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), to determine the abnormal CSF proteins in GBS patients. Our data showed that the levels of six proteins and their isoforms in CSF were significantly altered in GBS patients compared with controls. Haptoglobin, apolipoprotein A-IV and PRO2044 (unnamed protein) were considerably increased in the CSF of GBS patients, whereas transthyretin, apolipoprotein E and fibrinogen were considerably decreased. We concluded that these six proteins may be involved in the pathogenesis of GBS and call for further studying the role of these proteins in the pathogenesis of the disease. Show less

This study aims to identify the effects of antipsychotics on plasma proteins, and on the proteins associated with schizophrenia. We applied proteomics technology to screen protein aberrations in Sprague-Dawley rats treated with antipsychotics and schizophrenic patients undergoing medication. ApoA-I was found significantly increased in the chlorpromazine-treated rats and decreased in the patients with treatment-resistant schizophrenia, which suggest that decreased levels of apoA-I might be associated with the pathology of schizophrenia and that chlorpromazine increases apoA-I levels as part of its therapeutic action. Show less

To investigate the relationship between the polymorphism of apolipoprotein A5 gene (APOA5) -12238 T>C and atherosclerotic cerebral infarction (ACI). Three hundred and forty-one subjects (170 ACI patients and 171 healthy controls) were collected to determine the genotypes by using polymerase chain reaction-restriction fragment length polymorphisms. APOA5 allele frequencies of T/C were 0.588/0.412 and 0.424/0.576 in ACI group and control group respectively. There was significant difference in allele and genotype frequencies between ACI group and control group (P < 0.05). The levels of plasma triglyceride in ACI patients with TT genotype were higher than those in patients with CC genotypes (P < 0.05). The relationship is found between the site of APOA5 gene -12238 T>C and ACI. There is a significant correlation between TT genotype of APOA5 and the levels of plasma triglyceride in patients with ACI. Show less

The genetic determinants of severe hypertriglyceridemia (HTG; MIM 144650) in adults are poorly defined. We therefore resequenced 3 candidate genes, namely LPL, APOC2, and APOA5, to search for accumulation of missense mutations in patients with severe HTG compared with normolipidemic subjects. We resequenced >2 million base pairs of genomic DNA from 110 nondiabetic patients with severe HTG and determined the prevalence of coding sequence variants compared with 472 age- and sex-matched normolipidemic controls. We found: (1) heterozygous mutations (LPL p.Q-12E >11X, p.D25H, p.W86R, p.G188E, p.I194T and p.P207L; APOC2 p.K19T and IVS2-30G>A) in 10.0% of severe HTG patients compared with 0.2% of controls (carrier odds ratio [OR] 52, 95% confidence interval [CI] 8.6 to 319); and (2) an association of the APOA5 p.S19W missense variant with severe HTG (carrier OR 5.5 95% CI 3.3 to 9.1). Furthermore, either rare mutations or the APOA5 p.S19W variant were found in 41.8% of HTG subjects compared with 8.9% of controls (carrier OR 7.4, 95% CI 4.5 to 12.0). Also, heterozygotes for rare mutations had a significantly reduced plasma triglyceride response to fibrate monotherapy. Both common and rare DNA variants in candidate genes were found in a substantial proportion of severe HTG patients. The findings underscore the value of candidate gene resequencing to understand the genetic contribution in complex lipoprotein and metabolic disorders. Show less