👤 Li Ren

A Mediterranean diet increases intakes of n-3 and n-9 fatty acids and lowers intake of n-6 fatty acids. This can impact colon cancer risk as n-6 fatty acids are metabolized to proinflammatory eicosanoids. The purpose of this study was to evaluate interactions of polymorphisms in the fatty acid desaturase (FADS) genes, FADS1 and FADS2, and changes in diet on fatty acid concentrations in serum and colon. A total of 108 individuals at increased risk of colon cancer were randomized to either a Mediterranean or a Healthy Eating diet. Fatty acids were measured in both serum and colonic mucosa at baseline and after six months. Each individual was genotyped for four single-nucleotide polymorphisms in the FADS gene cluster. Linear regression was used to evaluate the effects of diet, genotype, and the diet by genotype interaction on fatty acid concentrations in serum and colon. Genetic variation in the FADS genes was strongly associated with baseline serum arachidonic acid (n-6) but serum eicosapentaenoic acid (n-3) and colonic fatty acid concentrations were not significantly associated with genotype. After intervention, there was a significant diet by genotype interaction for arachidonic acid concentrations in colon. Subjects who had all major alleles for FADS1/2 and were following a Mediterranean diet had 16% lower arachidonic acid concentrations in the colon after six months of intervention than subjects following the Healthy Eating diet. These results indicate that FADS genotype could modify the effects of changes in dietary fat intakes on arachidonic acid concentrations in the colon. Show less

We used a complete spinal cord transection model and locomotor function, histological, and immunohistochemical examinations to evaluate the effects of local injection of lentivirus/LINGO-1-short hairpin RNA (VL) on rats with spinal cord injury (SCI). To demonstrate the neuroregenerative and neuroprotective effects of LINGO-1 RNAi on complete transection SCI rats. LINGO-1 has been reported as a negative regulator of axonal sprouting and its antagonist was determined to improve functional outcomes in SCI rats. However, it has not been assessed whether blockade of LINGO-1 mediated by lentivirus vectors could stimulate neural recovery after SCI. Complete spinal cord transection was made at T10 level. Suspension of lentivirus vectors encoding LINGO-1-short hairpin RNA was injected into the lesion gap. Controls received control vectors in the same manner and the sham group was subjected to laminectomy only. The Basso-Beattie-Bresnahan scale and surface righting reflex test were used to evaluate functional outcomes. Finally, the spinal cords were harvested for histological and immunohistochemical analysis. The treatment with VL improved Basso-Beattie-Bresnahan scores and surface righting reflex after SCI. Tissue repair was facilitated and the cavity area was significantly decreased in VL-treated animals. More sprouting and myelinated nerve fibers were detected within the injured site in the VL group as compared with the control. In addition, the number of survival neurons and oligodendrocytes around the epicenter was notably higher under the VL condition. Local injection of lentivirus/LINGO-1-short hairpin RNA after complete transection of spinal cord resulted in meaningful histological and functional outcomes in rats. The mechanism of VL protection may be related to its promotion of axonal sprouting, remyelination, and cell survival. Show less

The expression changes of liver X receptor alpha (LXRα), histone deacetylase 3 (HDAC3) and CCAAT/enhancer binding protein alpha (C/EBPα) were detected in liver tissues of our high-fat-diet E3 rat model. The aim of this study is to pinpoint the molecular mechanism of HDAC3 and C/EBPα to orchestrate LXRα expression in hepatocytes. We confirmed that LXRα and its target genes were negatively regulated by HDAC3 in stable expressed clones with pEGFP-Hdac3 or shRNA-Hdac3 vector. However, transient pEGFP-C/EBPα plasmid transfection showed an upregulation of LXRα expression and C/EBPα enhanced LXRα promoter activity in a dose-dependent manner in CBRH-7919 cells. By using 5'-serial deletion reporter analysis, we identified that fragment from -2881 to -1181bp of LXRα promoter was responsible for C/EBPα binding to the promoter, especially CBS1 and CBS4 were identified essentially by using ChIP and luciferase reporter assay. Co-IP, qRT-PCR and ChIP revealed that HDAC3 interacted with C/EBPα co-regulated LXRα expression. Sumoylation of C/EBPα at lysine 159 was detected in CBRH-7919 cells with transient overexpressed C/EBPα, and Co-IP assay detected that sumoylated C/EBPα interacted with more HDAC3 than C/EBPα K159L mutant. Luciferase reporter assay demonstrated that C/EBPα participated in HDAC3-repressed LXRα transcription, and HDAC3 was involved in sumoylated C/EBPα-inactivated LXRα activity. Luciferase reporter assay demonstrated that sumoylation of C/EBPα by SUMO-1 directly reversed the activation of C/EBPα on LXRα promoter. The results suggested that HDAC3 interacts with sumoylated C/EBPα to negatively regulate the LXRα expression. Show less

CBX5, CBX1, and CBX3 (HP1α, β, and γ, respectively) play an evolutionarily conserved role in the formation and maintenance of heterochromatin. In addition, CBX5, CBX1, and CBX3 may also participate in transcriptional regulation of genes. Recently, CBX3 binding to the bodies of a subset of genes has been observed in human and murine cells. However, the generality of this phenomenon and the role CBX3 may play in this context are unknown. Genome-wide localization analysis reveals CBX3 binding at genic regions, which strongly correlates with gene activity across multiple cell types. Depletion of CBX3 resulted in down-regulation of a subset of target genes. Loss of CBX3 binding leads to a more dramatic accumulation of unspliced nascent transcripts. In addition, we observed defective recruitment of splicing factors, including SNRNP70, to CBX3 target genes. Collectively, our data suggest a role for CBX3 in aiding in efficient cotranscriptional RNA processing. Show less

Activation of MEK5 in many cancers is associated with carcinogenesis through aberrant cell proliferation. In this study, we determined the level of phosphorylated MEK5 (pMEK5) expression in human colorectal cancer (CRC) tissues and correlated it with clinicopathologic data. pMEK5 expression was examined by immunohistochemistry in a tissue microarray (TMA) containing 335 clinicopathologic characterized CRC cases and 80 cases of nontumor colorectal tissues. pMEK5 expression of 19 cases of primary CRC lesions and paired with normal mucosa was examined by Western blotting. The relationship between pMEK5 expression in CRC and clinicopathologic parameters, and the association of pMEK5 expression with CRC survival were analyzed respectively. pMEK5 expression was significantly higher in CRC tissues (185 out of 335, 55.2%) than in normal tissues (6 out of 80, 7.5%; P < 0.001). Western blotting demonstrated that pMEK5 expression was upregulated in 12 of 19 CRC tissues (62.1%) compared to the corresponding adjacent nontumor colorectal tissues. Overexpression of pMEK5 in CRC tissues was significantly correlated to the depth of invasion (P = 0.001), lymph node metastasis (P < 0.001), distant metastasis (P < 0.001) and high preoperative CEA level (P < 0.001). Consistently, the pMEK5 level in CRC tissues was increased following stage progression of the disease (P < 0.001). Analysis of the survival curves showed a significantly worse 5-year disease-free (P = 0.002) and 5-year overall survival rate (P < 0.001) for patients whose tumors overexpressed pMEK5. However, in multivariate analysis, pMEK5 was not an independent prognostic factor for CRC (DFS: P = 0.139; OS: P = 0.071). pMEK5 expression is correlated with the staging of CRC and its expression might be helpful to the TNM staging system of CRC. Show less

Cytosolic sulfotransferase (SULT2B1b) catalyzes oxysterol sulfation. 5-Cholesten-3β-25-diol-3-sulfate (25HC3S), one product of this reaction, decreases intracellular lipids in vitro by suppressing liver X receptor/sterol regulatory element binding protein (SREBP)-1c signaling, with regulatory properties opposite to those of its precursor 25-hydroxycholesterol. Upregulation of SULT2B1b may be an effective strategy to treat hyperlipidemia and hepatic steatosis. The objective of the study was to explore the effect and mechanism of oxysterol sulfation by SULT2B1b on lipid metabolism in vivo. C57BL/6 and LDLR(-/-) mice were fed with high-cholesterol diet or high-fat diet for 10 weeks and infected with adenovirus encoding SULT2B1b. SULT2B1b expressions in different tissues were determined by immunohistochemistry and Western blot. Sulfated oxysterols in liver were analyzed by high-pressure liquid chromatography. Serum and hepatic lipid levels were determined by kit reagents and hematoxylin and eosin staining. Gene expressions were determined by real-time reverse transcriptase polymerase chain reaction and Western Blot. Following infection, SULT2B1b was successfully overexpressed in the liver, aorta, and lung tissues, but not in the heart or kidney. SULT2B1b overexpression, combined with administration of 25-hydroxycholesterol, significantly increased the formation of 25HC3S in liver tissue and significantly decreased serum and hepatic lipid levels, including triglycerides, total cholesterol, free cholesterol, and free fatty acids, as compared with controls in both C57BL/6 and LDLR(-/-) mice. Gene expression analysis showed that increases in SULT2B1b expression were accompanied by reduction in key regulators and enzymes involved in lipid metabolism, including liver X receptor α, SREBP-1, SREBP-2, acetyl-CoA carboxylase-1, and fatty acid synthase. These findings support the hypothesis that 25HC3S is an important endogenous regulator of lipid biosynthesis. Show less

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is regulated by endocytosis where many Rab proteins play an important role in the determination of the receptor recycle or degradation. In an effort to better understand how EGF signaling is regulated, we examined the role of Rab21 in regulation of the degradation and signal transduction of the EGFR. Using a transient expression protocol in HEK293T and HeLa cells, we found that Rab21 enhanced the degradation of EGFR through accelerating its internalization in both EGF-independent and EGF-dependent manners. We further demonstrated that Rab21 interacted with EGFR by immunoprecipitation experiments. Interestingly, we observed that overexpression of Rab21 attenuated EGF-mediated mitogen-activated protein kinase (MAPK) signaling by inducing EGFR degradation. Taken together, these data suggest that Rab21 plays a negative role in the EGF-mediated MAPK signaling pathway. Show less

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

The ESCRT-0 and ESCRT-I complexes coordinate the clustering of ubiquitinated cargo with intralumenal budding of the endosomal membrane, two essential steps in vacuolar/lysosomal protein sorting from yeast to humans. The 1.85-Å crystal structure of interacting regions of the yeast ESCRT-0 and ESCRT-I complexes reveals that PSDP motifs of the Vps27 ESCRT-0 subunit bind to a novel electropositive N-terminal site on the UEV domain of the ESCRT-I subunit Vps23 centred on Trp16. This novel site is completely different from the C-terminal part of the human UEV domain that binds to P(S/T)AP motifs of human ESCRT-0 and HIV-1 Gag. Disruption of the novel PSDP-binding site eliminates the interaction in vitro and blocks enrichment of Vps23 in endosome-related class E compartments in yeast cells. However, this site is non-essential for sorting of the ESCRT cargo Cps1. Taken together, these results show how a conserved motif/domain pair can evolve to use strikingly different binding modes in different organisms. Show less

In the previous experiment, we found that there was a different response between E3 rats and DA.1U rats to high-fat-diet-induced metabolic syndrome (HFD-MetS). The aim of this study was to explore the cause and molecular mechanism of the genetic difference in susceptibility to metabolic syndrome in E3 rats as compared with DA.1U rats. Firstly, a 12-week HFD-MetS model in E3 and DA.1U rats was carried out and assessed. Then, the expression of key insulin signaling molecules, metabolic nuclear receptors, metabolic key enzymes and histone deacetylases (Hdacs) was determined by different methods. Finally, the effects of overexpression and disruption of Hdac3 on metabolic nuclear receptors were analyzed in CBRH-7919 cells and primarily-hepatic cells from DA.1U and E3 rats. We found that E3 rats were susceptible, while DA.1U rats were resisted to HFD-MetS. The expression of liver X receptor α,β (LXR-α,β), farnesoid X receptor (FXR), peroxisome proliferator activated receptor γ (PPAR-γ) and cholesterol 7α-hydroxylase (CYP7A1) increased markedly in DA.1U rat liver, whereas they decreased significantly in E3 rats. The expression of Hdac3 increased by HFD treatment in both E3 and DA.1U rat livers, but the constitutive Hdac3 expression was lower in DA.IU rat liver than in E3 rat liver. Importantly, overexpression of Hdac3 could downregulate the expression of LXR-α, PPAR-γ and CYP7A1 in both CBRH-7919 cells and primarily cultured hepatic cells from DA.IU rats. On the contrary, disruption of Hdac3 by shRNA upregulated the expression of LXR-α, PPAR-γ and CYP7A1 in both CBRH-7919 cells and primarily cultured hepatic cells from E3 rats. The results suggested that a high constitutive expression of Hdac3 inhibiting the expression of PPAR-γ, LXR-α and CYP7A1 in liver contributes to HFD-MetS in E3 rats. Show less

Extracellular K⁺, Na⁺, and Ca²⁺ ions all influence the resting membrane potential of the neuron. However, the mechanisms by which extracellular Na⁺ and Ca²⁺ regulate basal neuronal excitability are not well understood. Recent findings suggest that NALCN, in association with UNC79 and UNC80, contributes a basal Na⁺ leak conductance in neurons. Mutations in Nalcn, Unc79, or Unc80 lead to severe phenotypes that include neonatal lethality and disruption in rhythmic behaviors. This review discusses the properties of the NALCN complex, its regulation, and its contribution to neuronal function and animal behavior. Show less

VHS (Vps27, Hrs, and STAM) domains occur in ESCRT-0 subunits Hrs and STAM, GGA adapters, and other trafficking proteins. The structure of the STAM VHS domain-ubiquitin complex was solved at 2.6 A resolution, revealing that determinants for ubiquitin recognition are conserved in nearly all VHS domains. VHS domains from all classes of VHS-domain containing proteins in yeast and humans, including both subunits of ESCRT-0, bound ubiquitin in vitro. ESCRTs have been implicated in the sorting of Lys63-linked polyubiquitinated cargo. Intact human ESCRT-0 binds Lys63-linked tetraubiquitin 50-fold more tightly than monoubiquitin, though only 2-fold more tightly than Lys48-linked tetraubiquitin. The gain in affinity is attributed to the cooperation of flexibly connected VHS and UIM motifs of ESCRT-0 in avid binding to the polyubiquitin chain. Mutational analysis of all the five ubiquitin-binding sites in yeast ESCRT-0 shows that cooperation between them is required for the sorting of the Lys63-linked polyubiquitinated cargo Cps1 to the vacuole. Show less

The liver X receptor (LXR) and constitutive androstane receptor (CAR) are two nuclear receptors postulated to have distinct functions. LXR is a sterol sensor that promotes lipogenesis, whereas CAR is a xenosensor that controls xenobiotic responses. Here, we show that LXRα and CAR are functionally related in vivo. Loss of CAR increased the expression of lipogenic LXR target genes, leading to increased hepatic triglyceride accumulation, whereas activation of CAR inhibited the expression of LXR target genes and LXR ligand-induced lipogenesis. On the other hand, a combined loss of LXR α and β increased the basal expression of xenobiotic CAR target genes, whereas activation of LXR inhibited the expression of CAR target genes and sensitized mice to xenobiotic toxicants. The mutual suppression between LXRα and CAR was also observed in cell culture and reporter gene assays. LXRα, like CAR, exhibited constitutive activity in the absence of an exogenously added ligand by recruiting nuclear receptor coactivators. Interestingly, although CAR competed with LXRα for coactivators, the constitutive activity and recruitment of coactivators was not required for CAR to suppress the activity of LXRα. In vivo chromatin immunoprecipitation assay showed that cotreatment of a CAR agonist compromised the LXR agonist responsive recruitment of LXRα to Srebp-1c, whereas an LXR agonist inhibited the CAR agonist-responsive recruitment of CAR to Cyp2b10. In conclusion, our results have revealed dual functions of LXRα and CAR in lipogenesis and xenobiotic responses, establishing a unique role of these two receptors in integrating xenobiotic and endobiotic homeostasis. Show less

In contrast to its extensively studied intracellular roles, the molecular mechanisms by which extracellular Ca(2+) regulates the basal excitability of neurons are unclear. One mechanism is believed to be through Ca(2+)'s interaction with the negative charges on the cell membrane (the charge screening effect). Here we show that, in cultured hippocampal neurons, lowering [Ca(2+)](e) activates a NALCN channel-dependent Na(+)-leak current (I(L-Na)). The coupling between [Ca(2+)](e) and NALCN requires a Ca(2+)-sensing G protein-coupled receptor, an activation of G-proteins, an UNC80 protein that bridges NALCN to a large novel protein UNC79 in the same complex, and the last amino acid of NALCN's intracellular tail. In neurons from nalcn and unc79 knockout mice, I(L-Na) is insensitive to changes in [Ca(2+)](e), and reducing [Ca(2+)](e) fails to elicit the excitatory effects seen in the wild-type. Therefore, extracellular Ca(2+) influences neuronal excitability through the UNC79-UNC80-NALCN complex in a G protein-dependent fashion. Show less

The significance of transcription factors PPAR alpha, LXR alpha, and their responsive/target genes for the pathogenesis of atherosclerosis in apolipoprotein E and low-density lipoprotein receptor double deficient (AL) mice fed with high fat and cholesterol (HF) diet were studied. C57BL/6J wild-type (WT) mice were used as control to the AL mice. Plasma lipid metabolites and morphological atherosclerotic lesions in aortic wall were determined. Semi- and real-time quantitative RT-PCR were used to measure gene expression patterns between AL mice and the controls, which were fed with HF or normal chow diet. The results showed that in AL mice fed with HF diet, plasma lipid levels, hepatic lipid accumulation, and atherogenesis together with upregulated PPAR alpha, LXR alpha, and their target genes, i.e., FAT, SCD1, FAS, Angptl3, and apoB100 significantly increased in a 12-week long feeding period. In contrast, apoAI, apoAIV, apoF, LPL, and SR-BI were decreased compared to chow-fed group. In WT mice, PPAR alpha, LXR alpha, FAS, Angpt13, CPT1, apoF, ACOX1, LPL, and SR-BI were increased with HF treatment, while apoAI and apoAIV were decreased markedly. The different changes of lipid metabolism-related genes between AL and WT mice, fed with HF diet or chow diet indicated that the mechanisms of dietary effects on gene mutant mice are different from those of intact WT mice. Since lipid metabolic system defected genetically in AL mice, we suggest that the changes of PPAR alpha, LXR alpha, and their target genes aggravated lipid metabolic disorder in the liver and further accelerated the development of atherosclerosis on a stress of HF diet feeding in AL mice. Show less

Hyperlipidemia is one of the most important risk factors for atherosclerosis. This can be amplified by a localized inflammatory response mediated by macrophages. Macrophages are capable of taking up excess cholesterol, and it is well known that delivery of cholesterol to the mitochondria by steroidogenic acute regulatory (StAR) protein is the rate-limiting step for cholesterol degradation in the liver. It has also been shown that overexpression of StAR in hepatocytes dramatically increases the amount of regulatory oxysterols in the nucleus, which play an important role in the maintenance of intracellular lipid homeostasis. The goal of the present study was to determine whether StAR plays a similar role in macrophages. We have found that overexpression of StAR in human THP-1 monocyte-derived macrophages decreases intracellular lipid levels, activates liver X receptor alpha (LXRalpha) and proliferation peroxysome activator receptor gamma (PPARgamma), and increases ABCG1 and CYP27A1 expression. Furthermore, it reduces the secretion of inflammatory factors, and prevents apoptosis. These results suggest that StAR delivers cholesterol to mitochondria where regulatory oxysterols are generated. Regulatory oxysterols can in turn activate nuclear receptors, which increase expression of cholesterol efflux transporters, and decrease secretion of inflammatory factors. These effects can prevent macrophage apoptosis. These results imply a potential role of StAR in the prevention of atherosclerosis. Show less

Ubiquitin (Ub) is a sorting signal that targets integral membrane proteins to the interior of the vacuole/lysosome by directing them into lumenal vesicles of multivesicular bodies (MVBs). The Vps27-Hse1 complex, which is homologous to the Hrs-STAM complex in mammalian cells, serves as a Ub-sorting receptor at the surface of early endosomes. We have found that Hse1 interacts with Doa1/Ufd3. Doa1 is known to interact with Cdc48/p97 and Ub and is required for maintaining Ub levels. We find that the Hse1 Src homology 3 domain binds directly to the central PFU domain of Doa1. Mutations in Doa1 that block Hse1 binding but not Ub binding do not alter Ub levels but do result in the missorting of the MVB cargo GFP-Cps1. Loss of Doa1 also causes a synthetic growth defect when combined with loss of Vps27. Unlike the loss of Doa1 alone, the doa1Delta vps27Delta double mutant phenotype is not suppressed by Ub overexpression, demonstrating that the effect is not due to indirect consequence of lowered Ub levels. Loss of Doa1 results in a defect in the accumulation of GFP-Ub within yeast vacuoles, implying that there is a reduction in the flux of ubiquitinated membrane proteins through the MVB pathway. This defect was also reflected by an inability to properly sort Vph1-GFP-Ub, a modified subunit of the multiprotein vacuolar ATPase complex, which carries an in-frame fusion of Ub as an MVB sorting signal. These results reveal novel roles for Doa1 in helping to process ubiquitinated membrane proteins for sorting into MVBs. Show less

Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known "xenobiotic receptor," could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) gamma were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRalpha. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARgamma, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARgamma. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARgamma regulation are liver specific. We conclude that Cd36 is a shared target of LXR, PXR, and PPARgamma. The network of CD36 regulation by LXR, PXR, and PPARgamma establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis. Show less

The retinoid-related orphan receptors (RORs) and liver X receptors (LXRs) were postulated to have distinct functions. RORs play a role in tissue development and circadian rhythm, whereas LXRs are sterol sensors that affect lipid homeostasis. In this study, we revealed a novel function of RORalpha (NR1F1) in regulating the oxysterol 7alpha-hydroxylase (Cyp7b1), an enzyme critical for the homeostasis of cholesterol, bile acids, and oxysterols. The expression of Cyp7b1 gene was suppressed in the RORalpha null (RORalpha(sg/sg)) mice, suggesting RORalpha as a positive regulator of Cyp7b1. Promoter analysis established Cyp7b1 as a transcriptional target of RORalpha, and transfection of RORalpha induced the expression of endogenous Cyp7b1 in the liver. Interestingly, Cyp7b1 regulation seemed to be RORalpha-specific, because RORgamma had little effect. Reporter gene analysis showed that the activation of Cyp7b1 gene promoter by RORalpha was suppressed by LXRalpha (NR1H3), whereas RORalpha inhibited both the constitutive and ligand-dependent activities of LXRalpha. The mutual suppression between RORalpha and LXR was supported by the in vivo observation that loss of RORalpha increased the expression of selected LXR target genes, leading to hepatic triglyceride accumulation. Likewise, mice deficient of LXR alpha and beta isoforms showed activation of selected RORalpha target genes. Our results have revealed a novel role for RORalpha and a functional interplay between RORalpha and LXR in regulating endo- and xenobiotic genes, which may have broad implications in metabolic homeostasis. Show less

Varp, a novel protein containing a VPS9 domain and ankyrin repeats, can function as a guanine nucleotide exchange factor (GEF) of Rab21. We previously reported that Varp plays an important role in the regulation of endosome dynamics. To further investigate the function of Varp, a yeast two-hybrid screen was performed and Rab38 was identified as a Varp-associated protein. We demonstrate that Varp physically interacts with Rab38, and preferentially binds to the active GTP-bound form of Rab38 both in vitro and in vivo. Furthermore, Varp was shown to be recruited to Rab38-positive organelles in an ankyrin-repeat 1 (ANK1)-dependent manner. Our data demonstrate that Varp is a potential effector of Rab38. Together with our previous study, we propose Varp serves as both an effector and a GEF by interacting with different Rabs in mammalian cells. Show less

Although oligonucleotide chips, cDNA microarrays, differential display reverse transcription-PCR, and other approaches have been used to screen implantation-related molecules, the mechanism by which embryo implantation occurs is still unknown. The aim of this study was to profile the differential gene expression between interimplantation site and implantation site in mouse uterus on day 5 of pregnancy by serial analysis of gene expression (SAGE). In our two SAGE libraries of 11-bp tags, the total numbers of tags sequenced were 48,121 for the interimplantation site and 50,227 for the implantation site. There were 1,039 tags specifically expressed at interimplantation site, and 1,252 tags specifically expressed at the implantation site. Based on the p value, there were 195 tags significantly up-regulated at the interimplantation site and 261 tags significantly up-regulated at the implantation site, of which 100 genes were single matched at the interimplantation site and 127 genes were single matched at the implantation site, respectively. By reverse transcription-PCR, the tag ratio between the implantation site and interimplantation site was verified on 14 significantly changed genes. Using in situ hybridization, 1810014L12Rik, Psmb5, Cd63, Npm1, Fads3, and Tagln2 were shown to be highly expressed at the implantation site compared with the interimplantation site. Compared with the interimplantation site, Ddx39 was strongly expressed in the subluminal stromal cells at the implantation site on day 5 of pregnancy. Ddx39 expression at the implantation site was specifically induced by active blastocysts. Additionally, Ddx39 expression was significantly up-regulated by estrogen in the ovariectomized mice. In our SAGE data, many implantation-related genes were identified in mouse uterus. Our data could be a valuable source for future study on embryo implantation. Show less

Schizosaccharomyces pombe Dim1p is required for maintaining the steady-state level of the anaphase-promoting complex or cyclosome (APC/C) component Lid1p and thus for maintaining the steady-state level and activity of the APC/C. To gain further insight into Dim1p function, we have investigated the mechanism whereby Dim1p influences Lid1p levels. We show that S. pombe cells lacking Dim1p or Saccharomyces cerevisiae cells lacking its ortholog, Dib1p, are defective in generalized pre-mRNA splicing in vivo, a result consistent with the identification of Dim1p as a component of the purified yeast U4/U6.U5 tri-snRNP complex. Moreover, we find that Dim1p is part of a complex with the splicing factor Prp1p. However, although Dim1p is required for efficient splicing of lid1(+) pre-mRNA, circumventing the necessity for this particular function of Dim1p is insufficient for restoring normal Lid1p levels. Finally, we provide evidence that Dim1p also participates in the nuclear export of lid1(+) mRNA and that it is likely the combined loss of both of these two Dim1p functions which compromises Lid1p levels in the absence of proper Dim1p function. These data indicate that a mechanism acting at the level of mRNA impacts the functioning of the APC/C, a critical complex in controlling mitotic progression. Show less

Several independent population studies have reported that the apolipoprotein C3 (APOC3) Sst I polymorphism in apolipoprotein (apo) A1 /C3/A4/A5 gene cluster is associated with Hypertriglyceridaemia (HTG). HTG is a known risk factor for coronary atherosclerotic heart disease(CHD)and type II diabetes mellitus (non-insulin-dependent diabetes, NIDDM). The aim of this study is to investigate the association between the APOC3 gene Sst I polymorphism and the hypertriglyceridaemia in CHD and NIDDM in Chinese population. The genotype and allele frequencies of APOC3 Sst I polymorphism (S1/S2) were analyzed by PCR-restriction fragment length polymorphism in 267 CHD patients, 246 NIDDM patients and 491 unrelated healthy control individuals. The frequencies of minor allele 52 in CHD group, NIDDM group and control group were 0.301, 0.307 and 0.286, respectively. Compared with controls, there was no significant difference in distribution of genotype and allele frequencies of Sst I polymorphic site in CHD patients and NIDDM patients, respectively. However, the frequency of S1 S2 genotype in the HTG subgroup was significantly higher than that of the normal triglyceridaemia subgroup (NTG) in CHD patients (0.542 > 0.357, chi2 = 8.77, P = 0.0124). In NIDDM patients, the frequency of S2 S2 genotype in the HTG subgroup was significantly high, compared with that in the NTG subgroup (0.200 > 0.055, chi2 = 20.21, P = 0.0000), and there was significantly difference in the distribution of allele frequencies in subgroups of NTG and HTG (chi2 = 19.86, P = 0.0000). The level of triglyceride (TG) in S1 S2 genotype patients of CHD group were higher than that of S1 S1 genotype patients (P = 0.036). In NIDDM and controls groups, S2 S2 genotype individuals exhibited a significant increase in plasma TG concentrations, respectively compared with S1 S1 and S1 S2 genotype individuals of each group (P < 0.01). The minor allele S2, which was associated with both CHD with HTG and NIDDM with HTG and may contribute to the susceptibility of hypertriglyceridemia in CHD and NIDDM patients, may be one of the genetic predispositions to both CHD with HTG and NIDDM with HTG in Chinese population. Show less