👤 Junwei Shi

Reactive oxygen species (ROS) have been commonly accepted as inducers of autophagy, and autophagy in turn is activated to relieve oxidative stress. Yet, whether and how oxidative stress, generated in various human pathologies, regulates autophagy remains unknown. Here, we mechanistically studied the role of TRPM2 (transient receptor potential cation channel subfamily M member 2)-mediated Ca(2+) influx in oxidative stress-mediated autophagy regulation. On the one hand, we demonstrated that oxidative stress triggered TRPM2-dependent Ca(2+) influx to inhibit the induction of early autophagy, which renders cells more susceptible to death. On the other hand, oxidative stress induced autophagy (and not cell death) in the absence of the TRPM2-mediated Ca(2+) influx. Moreover, in response to oxidative stress, TRPM2-mediated Ca(2+) influx activated CAMK2 (calcium/calmodulin dependent protein kinase II) at levels of both phosphorylation and oxidation, and the activated CAMK2 subsequently phosphorylated BECN1/Beclin 1 on Ser295. Ser295 phosphorylation of BECN1 in turn decreased the association between BECN1 and PIK3C3/VPS34, but induced binding between BECN1 and BCL2. Clinically, acetaminophen (APAP) overdose is the most common cause of acute liver failure worldwide. We demonstrated that APAP overdose also activated ROS-TRPM2-CAMK2-BECN1 signaling to suppress autophagy, thereby causing primary hepatocytes to be more vulnerable to death. Inhibiting the TRPM2-Ca(2+)-CAMK2 cascade significantly mitigated APAP-induced liver injury. In summary, our data clearly demonstrate that oxidative stress activates the TRPM2-Ca(2+)-CAMK2 cascade to phosphorylate BECN1 resulting in autophagy inhibition. Show less

Individuals with dyslipidemia often develop type 2 diabetes, and diabetic patients often have dyslipidemia. It remains to be determined whether there are genetic connections between the 2 disorders. A female F2 cohort, generated from BALB/cJ (BALB) and SM/J (SM) Apoe-deficient (Apoe(-/-)) strains, was started on a Western diet at 6 weeks of age and maintained on the diet for 12 weeks. Fasting plasma glucose and lipid levels were measured before and after 12 weeks of Western diet. 144 genetic markers across the entire genome were used for quantitative trait locus (QTL) analysis. One significant QTL on chromosome 9, named Bglu17 [26.4 cM, logarithm of odds ratio (LOD): 5.4], and 3 suggestive QTLs were identified for fasting glucose levels. The suggestive QTL near the proximal end of chromosome 9 (2.4 cM, LOD: 3.12) was replicated at both time points and named Bglu16. Bglu17 coincided with a significant QTL for HDL (high-density lipoprotein) and a suggestive QTL for non-HDL cholesterol levels. Plasma glucose levels were inversely correlated with HDL but positively correlated with non-HDL cholesterol levels in F2 mice on either chow or Western diet. A significant correlation between fasting glucose and triglyceride levels was also observed on the Western diet. Haplotype analysis revealed that "lipid genes" Sik3, Apoa1, and Apoc3 were probable candidates for Bglu17. We have identified multiple QTLs for fasting glucose and lipid levels. The colocalization of QTLs for both phenotypes and the sharing of potential candidate genes demonstrate genetic connections between dyslipidemia and type 2 diabetes. Show less

Liver mitochondria affected by drugs can be released into circulation and serve as biomarkers for drug-induced liver injury (DILI). The tissue specificity of ALT was improved by differentiating cytosolic ALT1 and mitochondrial ALT2 isoforms released in circulation. Prior to ALT elevation, mitochondrial cytochrome c, OCT, GLDH, CPS1 and DNA were increased in circulation following DILI. The baseline expression of mt-Nd6 was predictive of individual DILI susceptibility in animals. As mitochondrial DILI biomarkers appeared to be drug or species dependent, they might have value in clinical scenarios when culprit drugs are established, but may not be ideal tools to assess DILI potentials of new drugs. Show less

N-acetylglutamate synthase (NAGS) catalyzes the production of N-acetylglutamate (NAG) from acetyl-CoA and L-glutamate. In microorganisms and plants, the enzyme functions in the arginine biosynthetic pathway, while in mammals, its major role is to produce the essential co-factor of carbamoyl phosphate synthetase 1 (CPS1) in the urea cycle. Recent work has shown that several different genes encode enzymes that can catalyze NAG formation. A bifunctional enzyme was identified in certain bacteria, which catalyzes both NAGS and N-acetylglutamate kinase (NAGK) activities, the first two steps of the arginine biosynthetic pathway. Interestingly, these bifunctional enzymes have higher sequence similarity to vertebrate NAGS than those of the classical (mono-functional) bacterial NAGS. Solving the structures for both classical bacterial NAGS and bifunctional vertebrate-like NAGS/K has advanced our insight into the regulation and catalytic mechanisms of NAGS, and the evolutionary relationship between the two NAGS groups. Show less

G-quadruplex (G4) DNA and G4 DNA resolvase are involved in a variety of biological processes. To understand the biological function of G4 DNA structures and their resolvases in spermatogenesis, we investigated the distribution of G4 structures in mouse testis and identified their alterations during spermatogenesis. Meanwhile, we studied the function of RNA helicase associated with AU-rich element (RHAU), a G4 DNA resolvase, in spermatogenesis with a germ-cell-specific knockout mouse model. The results showed that the ablation of RHAU in germ cells caused the increase of G4 structures and thus resulted in the decrease of spermatogonial differentiation. c-kit, a spermatogonia differentiation-related gene, contains two G4 DNA motifs on its promoter. We found its expression was significantly downregulated in RHAU conditional knockout testis. A further analysis demonstrated that RHAU directly bound to the G4 structures to activate c-kit expression. We concluded that RHAU regulates spermatogonia differentiation by promoting c-kit expression via directly binding to the G4 DNA motifs c-kit promoter. Show less

T cells play a crucial role in viral clearance or persistence; however, the precise mechanisms that control their responses during viral infection remain incompletely understood. MicroRNA (miR) has been implicated as a key regulator controlling diverse biological processes through posttranscriptional repression. Here, we demonstrate that hepatitis C virus (HCV)-mediated decline of miR-181a expression impairs CD4(+) T-cell responses through overexpression of dual specific phosphatase 6 (DUSP6). Specifically, a significant decline of miR-181a expression along with overexpression of DUSP6 was observed in CD4(+) T cells from chronically HCV-infected individuals compared to healthy subjects, and the levels of miR-181a loss were found to be negatively associated with the levels of DUSP6 overexpression in these cells. Importantly, reconstitution of miR-181a or blockade of DUSP6 expression in CD4(+) T cells led to improved T-cell responses including enhanced CD25 and CD69 expression, increased interleukin-2 expression, and improved proliferation of CD4(+) T cells derived from chronically HCV-infected individuals. Since a decline of miR-181a concomitant with DUSP6 overexpression is the signature marker for age-associated T-cell senescence, these findings provide novel mechanistic insights into HCV-mediated premature T-cell aging through miR-181a-regulated DUSP6 signaling and reveal new targets for therapeutic rejuvenation of impaired T-cell responses during chronic viral infection. Show less

Previous studies reported that miR-1908 was highly expressed in mature human adipocytes. Adipokines tumor necrosis factor α (TNF-α) that was known to activate NF-kappaB signaling could affect the expression of miR-1908 in adipocytes. However, little is known about the underlying mechanisms. In this study, we identified miR-1908 promoter using polymerase chain reaction (PCR) from human genomic DNA. Bioinformatic analysis was applied to predict the NF-kappaB binding sites in miR-1908 promoter. Real-time PCR, dual luciferase reporter assay, Mutagenesis analysis and electrophoretic mobility shift assay (EMSA) were performed to demonstrate the function of NF-kappaB binding sites in miR-1908 promoter. 1243bp miR-1908 promoter located in the intron of host gene fatty acid desaturase 1 (FADS1). Bioinformatic analysis revealed the presence of two putative NF-kappaB binding sites. TNF-α restricts miR-1908 expression in preadipocytes, and TNF-α decreases miR-1908 promoter activity in HEK293T cells. In addition, those two NF-kappaB transcription factor binding sites in miR-1908 promoter were functional. Our findings demonstrated that miR-1908 has its own transcription unit, and revealed the transcriptional mechanisms of miR-1908 expression based on NF-kappaB signaling. This study offers a theoretical basis for understanding the transcriptional mechanism of miR-1908 expression and may provide a new strategy for obesity clinical therapy. Show less

Several studies have shown associations between the composition of polyunsaturated fatty acids (PUFAs) in various tissues and type 2 diabetes mellitus (T2DM) development in European populations. Genetic variants of fatty acid desaturase (FADS) contribute to the variations of PUFA composition. Here we have explored whether similar correlations are also true among Chinese Han people. A case-control study was employed to examine this correlation in Han Chinese people. The study included 421 healthy adults and 331 T2DM patients. The ratio of arachidonic acid/linoleic acid (AA/LA), which reflects Δ6 desaturase activity, was significantly increased in T2DM patients. Furthermore, the ratio of eicosapentaenoic acid/α-linolenic acid (EPA/ALA), which reflects Δ5 desaturase activity, was markedly decreased in T2DM patients. Importantly, among four single nucleotide polymorphisms (rs174545, rs2072114, rs174602 and rs174616) in the FADS1-FADS2 gene cluster, only minor allele (T) of rs174616 was associated with decreased risk of T2DM in both codominant and dominant models after adjustment for age, gender and BMI. Furthermore, the ratio of AA/LA in both controls and T2DM was reduced in T carriers while an increased proportion of LA was seen in T2DM patients compared with control patients. These data suggest that in northern Han Chinese people, the minor allele (T) of rs174616 in the FADS1-FADS2 gene cluster is associated with a decreased conversion rate of LA to AA, which may contribute to decreased reduced risk of developing T2DM. Show less

In the present study, we demonstrate that prolonged treatment by trastuzumab induced resistance of NCI-N87 gastric cancer cells to trastuzumab. The resistant cells possessed typical characteristics of epithelial to mesenchymal transition (EMT)/cancer stem cells and acquired more invasive and metastatic potentials both in vitro and in vivo. Long term treatment with trastuzumab dramatically inhibited the phosphorylation of Akt, but triggered the activation of STAT3. The level of IL-6 was remarkably increased, implicating that the release of IL-6 that drives the STAT3 activation initiates the survival signaling transition. Furthermore, the Notch activities were significantly enhanced in the resistant cells, companied by upregulation of the Notch ligand Jagged-1 and the Notch responsive genes Hey1 and Hey2. Inhibiting the endogenous Notch pathway reduced the IL-6 expression and restored the sensitivities of the resistant cells to trastuzumab. Blocking of the STAT3 signaling abrogated IL-6-induced Jagged-1 expression, effectively inhibited the growth of the trastuzumab resistant cells, and enhanced the anti-tumor activities of trastuzumab in the resistant cells. These findings implicate that the IL-6/STAT3/Jagged-1/Notch axis may be a useful target and that combination of the Notch or STAT3 inhibitors with trastuzumab may prevent or delay clinical resistance and improve the efficacy of trastuzumab in gastric cancer. Show less

RUNX1-RUNX1T1 (formerly AML1-ETO), a transcription factor generated by the t(8;21) translocation in acute myeloid leukemia (AML), dictates a leukemic program by increasing self-renewal and inhibiting differentiation. Here we demonstrate that the histone demethylase JMJD1C functions as a coactivator for RUNX1-RUNX1T1 and is required for its transcriptional program. JMJD1C is directly recruited by RUNX1-RUNX1T1 to its target genes and regulates their expression by maintaining low H3K9 dimethyl (H3K9me2) levels. Analyses in JMJD1C knockout mice also establish a JMJD1C requirement for RUNX1-RUNX1T1's ability to increase proliferation. We also show a critical role for JMJD1C in the survival of multiple human AML cell lines, suggesting that it is required for leukemic programs in different AML cell types through its association with key transcription factors. Show less

The dysregulation of cholesterol metabolism and inflammation plays a significant role in the progression of diabetic nephropathy (DN). Anthocyanins are polyphenols widely distributed in food and exert various biological effects including antioxidative, anti-inflammatory, and antihyperlipidemic effects. However, it remains unclear whether anthocyanins are associated with DN, and the mechanisms involved in the reciprocal regulation of inflammation and cholesterol efflux are yet to be elucidated. In this study, we evaluated the regulation of cholesterol metabolism and the anti-inflammatory effects exerted by anthocyanins (cyanidin-3-O-β-glucoside chloride [C3G] or cyanidin chloride [Cy]) and investigated the underlying molecular mechanism of action using high-glucose (HG)-stimulated HK-2 cells. We found that anthocyanins enhanced cholesterol efflux and ABCA1 expression markedly in HK-2 cells. In addition, they increased peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptor alpha (LXRα) expression and decreased the HG-induced expression of the proinflammatory cytokines intercellular adhesion molecule-1 (ICAM1), monocyte chemoattractant protein-1 (MCP1), and transforming growth factor-β1 (TGFβ1), as well as NFκB activation. Incubation with the PPARα-specific inhibitor GW6471 and LXRα shRNA attenuated the anthocyanin-mediated promotion of ABCA1 expression and cholesterol efflux, suggesting that anthocyanins activated PPARα-LXRα-ABCA1-dependent cholesterol efflux in HK-2 cells. Moreover, the knockout of LXRα abrogated the anti-inflammatory effect of anthocyanins, whereas the PPARα antagonist GW6471 does not have this effect. Further investigations revealed that LXRα might interfere with anthocyanin-induced decreased ICAM1, MCP1, and TGFβ1 expression by reducing the nuclear translocation of NFκB. Collectively, these findings suggest that blocking cholesterol deposition and inhibiting the LXRα pathway-induced inflammatory response might be one of the main mechanisms by which anthocyanins exert their protective effects in DN. Show less

We studied the effects of alfalfa saponin extract (ASE) on low density lipoprotein receptor (Ldlr), liver X receptor α (LXRα), and farnesoid X receptor (FXR) in normal and hyperlipidemic Buffalo rat liver (BRL) cells. Normal and hyperlipidemic BRL cells were divided into eight groups: normal, or normal cells treated with 50, 100, and 150 mg/L ASE, hyperlipidemic, or hyperlipidemic cells treated with 50, 100, and 150 mg/L ASE. After treatment for 24 h, Ldlr, LXRα, and FXR mRNA expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Data showed that mRNA expression of Ldlr in normal BRL cells was significantly up-regulated by ASE treatment and mRNA expressions of LXRα and FXR were significantly down-regulated both in normal and hyperlipidemic BRL cells after ASE treatment. Thus, ASE might ameliorate hepatic steatosis by regulating genes involved in cholesterol metabolism, including up-regulation of Ldlr as well as down-regulation of LXRα and FXR. Show less

Cytoplasmic poly(A) binding protein 4 (PABPC4) is an RNA-processing protein which has an important role in regulating gene expression. The association of the PABPC4 rs4660293 single nucleotide polymorphism (SNP) and serum lipid profiles has, to the best of our knowledge, not previously been studied in the Chinese population. The present study aimed to investigate the association between the PABPC4 rs4660293 SNP and several environmental factors with serum lipid levels in the Mulao and Han populations. A total of 727 individuals of Mulao nationality and 729 individuals of Han nationality were randomly selected from stratified randomized samples from a previous study by our group. Genotypes of the PABPC4 rs4660293 SNP were determined via polymerase chain reaction and restriction fragment length polymorphism analyses and subsequently confirmed by direct sequencing. Serum levels of low-density lipoprotein cholesterol (LDL-C) and apolipoprotein (Apo) B were higher in the Mulao group than those in the Han group (P<0.01 for each). The genotypic and allelic frequencies of the PABPC4 rs4660293 SNP were significantly different between males and females in the Mulao population (P<0.05 for each), while no significant difference was detected between those of males and females amongst the Han population. The frequency of the G allele was higher in Mulao males than in Mulao females (22.12 vs. 13.44%). The G allele carriers were found to have higher total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and ApoAI levels in Han females but not in Han males, and lower TC and HDL-C levels in Mulao females but not in Mulao males than those of the G allele non-carriers (P<0.05 for all). These associations were confirmed by multiple linear regression analysis (P<0.05‑0.001). Serum lipid parameters were also correlated with multiple environmental factors (P<0.05‑0.001). The PABPC4 rs4660293 SNP was associated with serum TC, HDL-C, LDL-C and ApoAI levels in these study populations; however, the association varied between the Mulao and Han populations. A gender-specific association was identified in the populations of the two ethnic groups. Show less

Zinc finger protein 259 (ZNF259) binds to the cytoplasmic domain of epidermal growth factor receptor (EGFR) in quiescent cells and contributes tolipid metabolism. This case and control study investigated the association between ZNF259 single nucleotide polymorphisms (SNPs) and metabolic syndrome (MetS). This study included 1,812 MetS patients and 2,036 controls from the Jilin province of Northeastern China. MetS was diagnosed using the International Diabetes Federation (IDF) criteria. Three ZNF259 SNPs (rs964184, rs2075290 and rs2075294) were genotyped using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). There were significant differences between metabolic syndrome and healthy control subjects for the ZNF259 rs964184 and rs2075290 genotypes. The minor alleles of both SNPs were associated with an increased risk of MetS and associated conditions (elevated triglycerides, elevated blood pressure, increased abdominal obesity, fasting hyperglycemia, and low HDL-C; p < 0.05). The distribution of haplotype G-G-G (rs964184, rs2075290 and rs2075294) was significantly different between MetS patients and controls (OR = 1.39; 95% CI, 1.24 - 1.56; p < 0.01). This study demonstrated that ZNF259 variants were associated with elevated MetS risk in a Han Chinese population from the Jilin province of Northeastern China. Show less

Apolipoprotein A5 (APOA5) gene plays a key role in plasma triglyceride (TG) metabolism, and shows the involvement in coronary artery disease (CAD). A set of single nucleotide polymorphisms around the APOA5 gene was identified to be associated with plasma TG levels. It is of biological and clinical importance to discern the genuine genetic determinants. A polymorphism in 3' untranslated region of the APOA5 gene, rs2266788, is deserving of investigation for suggestive clues from the association in multiple independent studies. In this study, rs2266788 was genotyped in 3222 unrelated subjects consisting of 2062 CAD cases and 1160 controls. The statistical analyses indicated that the minor C allele of rs2266788 was significantly associated with elevated plasma TG levels and higher CAD risk. In normal human liver tissues, comparison of global APOA5 mRNA levels among genotypes and allelic expression imbalance analysis showed the decreased gene expression for the C allele. Luciferase assays confirmed a concordant result that transcriptional activity was lowered for the C allele compared with the T allele in four cell lines. Multiple lines of evidence in our study supported that rs2266788 was causally associated with plasma TG levels conferring CAD risk in Han Chinese population owing to a cis-acting effect to the APOA5 gene expression. Show less

To discuss the changes in Wnt pathway inhibiting factors in esophageal precancerosis lesions induced by methyl benzyl nitrosamine (MBNA) and the effect of Gexia Zhuyu decoction. Wistar rats were subcutaneously injected with MBNA (3.5 mg x kg(-1) for twice per week to establish the model. Since the 1st day after the model establishment, they were orally administered with Gexia Zhuyu decoction (16, 8 mg x kg(-1)). At the 10th week, esophageal tissues were collected to observe the pathological changes of esophageal mucosa, detect SFRP1, sFRP4, Axin1, Axin2 and GSK-3β mRNA levels.by fluorescent quantitation PCR analysis and β-catenin protein level by Western blotting. Being induced by MBNA, rats in the model group showed slight atypical hyperplasia in the histopathological examination. Compared with the normal group, Gexia Zhuyu decoction dose high and low groups showed no significant pathomorphological and histological changes. The model group showed lower gene transcription levels of esophageal tissues sFRP1, sFRP4, Axin1 and Axin2 (P < 0.05 or P < 0.01) and higher β-catenin protein expression level (P < 0.01) than the normal control group. The Gexia Zhuyu decoction low dose group showed higher gene transcription levels of esophageal tissues sFRP1, sFRP4, Axin1 and Axin2 (P < 0.05 or P < 0.01) and lower β-catenin protein expression level (P < 0.01) than the normal control group. Up-regulated β-catenin protein level and down-regulated Wnt pathway could enhance Wnt pathway activity of MBNA-induced esophageal precancerous lesions. Gexia Zhuyu decoction could down-regulate the β-catenin protein level and up-regulate the transcription level of Wnt pathway inhibiting factors, but could not block MBNA-induced esophageal precancerosis lesions. Show less

Known genetic loci explain only a small proportion of the familial relative risk of colorectal cancer (CRC). We conducted a genome-wide association study of CRC in East Asians with 14,963 cases and 31,945 controls and identified 6 new loci associated with CRC risk (P = 3.42 × 10(-8) to 9.22 × 10(-21)) at 10q22.3, 10q25.2, 11q12.2, 12p13.31, 17p13.3 and 19q13.2. Two of these loci map to genes (TCF7L2 and TGFB1) with established roles in colorectal tumorigenesis. Four other loci are located in or near genes involved in transcriptional regulation (ZMIZ1), genome maintenance (FEN1), fatty acid metabolism (FADS1 and FADS2), cancer cell motility and metastasis (CD9), and cell growth and differentiation (NXN). We also found suggestive evidence for three additional loci associated with CRC risk near genome-wide significance at 8q24.11, 10q21.1 and 10q24.2. Furthermore, we replicated 22 previously reported CRC-associated loci. Our study provides insights into the genetic basis of CRC and suggests the involvement of new biological pathways. Show less

Transforming growth factor-β (TGF-β) plays an important role in smooth muscle (SM) differentiation, but the downstream target genes regulating the differentiation process remain largely unknown. In this study, we identified olfactomedin 2 (Olfm2) as a novel regulator mediating SM differentiation. Olfm2 was induced during TGF-β-induced SM differentiation of human embryonic stem cell-derived mesenchymal cells. Olfm2 knockdown suppressed TGF-β-induced expression of SM markers, including SM α-actin, SM22α, and SM myosin heavy chain, whereas Olfm2 overexpression promoted the SM marker expression. TGF-β induced Olfm2 nuclear accumulation, suggesting that Olfm2 may be involved in transcriptional activation of SM markers. Indeed, Olfm2 regulated SM marker expression and promoter activity in a serum response factor (SRF)/CArG box-dependent manner. Olfm2 physically interacted with SRF without affecting SRF-myocardin interaction. Olfm2-SRF interaction promoted the dissociation of SRF from HERP1, a transcriptional repressor. Olfm2 also inhibited HERP1 expression. Moreover, blockade of Olfm2 expression inhibited TGF-β-induced SRF binding to SM gene promoters in a chromatin setting, whereas overexpression of Olfm2 dose dependently enhanced SRF binding. These results demonstrate that Olfm2 mediates TGF-β-induced SM gene transcription by empowering SRF binding to CArG box in SM gene promoters. Show less

Cysteine proteases play important roles in pathobiology. Here we reveal that cathepsin K (CatK) has a role in ischaemia-induced neovascularization. Femoral artery ligation-induced ischaemia in mice increases CatK expression and activity, and CatK-deficient mice show impaired functional recovery following hindlimb ischaemia. CatK deficiency reduces the levels of cleaved Notch1 (c-Notch1), Hes1 Hey1, Hey2, vascular endothelial growth factor, Flt-1 and phospho-Akt proteins of the ischaemic muscles. In endothelial cells, silencing of CatK mimicked, whereas CatK overexpression enhanced, the levels of c-Notch1 and the expression of Notch downstream signalling molecules, suggesting CatK contributes to Notch1 processing and activates downstream signalling. Moreover, CatK knockdown leads to defective endothelial cell invasion, proliferation and tube formation, and CatK deficiency is associated with decreased circulating endothelial progenitor cells-like CD31(+)/c-Kit(+) cells in mice following hindlimb ischaemia. Transplantation of bone marrow-derived mononuclear cells from CatK(+/+) mice restores the impairment of neovascularization in CatK(-/-) mice. We conclude that CatK may be a potential therapeutic target for ischaemic disease. Show less

Early life stress is one of the major susceptible factors for stress-related pathologies like posttraumatic stress disorder (PTSD). Recent studies in rats suggest that rather than being overall unfavorable, early life stress may prepare the organism to perform optimally to stressful environments later in life. In this study, severely adverse early life stress was conducted by six consecutive hours of maternal separation (MS), from PND1 to PND21, and contextual fear conditioning model was used on PND90 to mimic the second stress in adulthood and the re-experiencing symptom of PTSD. It was observed that in this investigation pups experienced MS showed decreased sensibility to contextual fear conditioning in adulthood, and there sex plays an important role. For example, female rats suffered MS had much lower freezing than males and controls. Meanwhile, Morris water maze test indicated that MS did not impair rat's performance of spatial learning and memory. Furthermore, suppression subtractive hybridization (SSH) was used to screen the related genes of fear memory, by examining the changes of mRNA expression in CA1 area between female MS and control rats after contextual fear conditioning. Finally, nine up-regulated and one down-regulated genes, including β2-MG, MAF, Nd1-L, TorsinA and MACF1 gene were found in this study. It is assumed that the TorsinA, MACF1 and Nd1-L gene may contribute to the decreased sensitivity of PTSD induced by MS. Show less

Inflammation marks all stages of atherogenesis. DNA hypermethylation in the whole genome or specific genes is associated with inflammation and cardiovascular diseases. Therefore, we aimed to study whether inhibiting DNA methylation by DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) ameliorates atherosclerosis in low-density lipoprotein receptor knockout (Ldlr(-/-)) mice. Ldlr(-/-) mice were fed an atherogenic diet and adminisered saline or 5-aza-dC (0.25 mg/kg) for up to 30 weeks. 5-aza-dC treatment markedly decreased atherosclerosis development in Ldlr(-/-) mice without changes in body weight, plasma lipid profile, macrophage cholesterol levels and plaque lipid content. Instead, this effect was associated with decreased macrophage inflammation. Macrophages with 5-aza-dC treatment had downregulated expression of genes involved in inflammation (TNF-α, IL-6, IL-1β, and inducible nitric oxidase) and chemotaxis (CD62/L-selectin, chemokine [C-C motif] ligand 2/MCP-1 [CCL2/MCP-1], CCL5, CCL9, and CCL2 receptor CCR2). This resulted in attenuated macrophage migration and adhesion to endothelial cells and reduced macrophage infiltration into atherosclerotic plaques. 5-aza-dC also suppressed macrophage endoplasmic reticulum stress, a key upstream signal that activates macrophage inflammation and apoptotic pathways. Finally, 5-aza-dC demethylated liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ1 (PPARγ1) promoters, which are both enriched with CpG sites. This led to overexpression of LXRα and PPARγ, which may be responsible for 5-aza-dC's anti-inflammatory and atheroprotective effect. Our findings provide strong evidence that DNA methylation may play a significant role in cardiovascular diseases and serve as a therapeutic target for prevention and treatment of atherosclerosis. Show less

This study was designed to investigate the direct effects of fatty acids (FAs) on the cell viability and the expression levels of genes involved in lipid metabolism in LO2 human liver cells. Palmitate (PA), oleate (OA) and docosahaexenoic acid (DHA) were used to represent saturated, mono-unsaturated and polyunsaturated FAs, respectively. At concentrations of ≤3.2 µg/ml, treatment with single FAs increased the viability of the LO2 cells. At FA concentrations of >3.2 µg/ml, cell viability following OA treatment was increased, but PA or DHA treatment at these concentrations reduced cell viability. Administration of mixtures of these FAs in three ratios (PA:OA:DHA = 1:2:1, 1:1:1 and 1:1:2, respectively) increased the cell viability compared with the control group. The intracellular triglyceride (TG) levels following all types of treatment were significantly increased and the accumulation of TGs was markedly increased with high doses of DHA. In addition, peroxisome proliferator-activated receptor-γ was significantly upregulated in all groups, with the exception of the 1:1:1 group at 3.2 µg/ml and the 1:1:2 group at 12.8 µg/ml. The expression levels of sterol regulatory-element binding protein‑1c, liver X receptor α and apolipoprotein C‑I were significantly reduced in all groups with the exception of the DHA‑treated group and the 1:2:1 groups at 3.2 and 12.8 µg/ml. In conclusion, these results indicate that the type, concentration and mixture ratios of FAs are all important in determining the cell viability and lipid metabolism-related gene expression in LO2 hepatocytes. Show less

In nonruminants, the alternative splicing of peroxisome proliferator-activated receptor γ (PPARG) generates PPARG1 and PPARG2 isoforms. Although transcriptional control differences between isoforms have been reported in human adipose tissue, their roles in ruminant mammary cells are not well known. To assess which of these isoforms is more closely associated with the regulation of mammary lipogenic pathways, their tissue distribution was analyzed and the expression of key genes regulating lipogenic gene networks was measured after overexpression of the 2 isoforms in goat mammary epithelial cells (GMEC). The expression of PPARG2 was markedly greater in adipose tissue, whereas PPARG1 is the main isoform in goat mammary tissue (ratio of PPARG1:PPARG2 was close to 37:1). As was reported in previous work, PPARG1 upregulated the transcription regulators SREBF1 and PPARG and the lipogenic genes FASN, ACACA, and SCD. Along with a tendency for greater expression of AGPAT6, DGAT1, and PLIN2, these data suggest that PPARG1 is the isoform controlling lipogenesis in mammary cells. Addition of the PPARG ligand rosiglitazone (ROSI) to GMEC overexpressing both isoforms upregulated the expression of LPL and CD36, which help control uptake of long-chain fatty acids into mammary cells. Other responses to ROSI addition to GMEC overexpressing PPARG1 and PPARG2 included upregulation of AGPAT6, DGAT1, INSIG1, SREBF1, and NR1H3. Although the data suggest that both PPARG1 and PPARG2 could affect mammary lipogenesis via control of gene expression when stimulated (e.g., by ROSI), the fact that PPARG1 is more abundant in mammary tissue and that its overexpression alone upregulated key lipogenic gene networks suggest that it is the more important isoform in goat mammary cells. Show less

In budding yeast cells, nutrient repletion induces rapid exit from quiescence and entry into a round of growth and division. The G1 cyclin CLN3 is one of the earliest genes activated in response to nutrient repletion. Subsequent to its activation, hundreds of cell-cycle genes can then be expressed, including the cyclins CLN1/2 and CLB5/6. Although much is known regarding how CLN3 functions to activate downstream targets, the mechanism through which nutrients activate CLN3 transcription in the first place remains poorly understood. Here we show that a central metabolite of glucose catabolism, acetyl-CoA, induces CLN3 transcription by promoting the acetylation of histones present in its regulatory region. Increased rates of acetyl-CoA synthesis enable the Gcn5p-containing Spt-Ada-Gcn5-acetyltransferase transcriptional coactivator complex to catalyze histone acetylation at the CLN3 locus alongside ribosomal and other growth genes to promote entry into the cell division cycle. Show less

N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K. Show less

To screen for potential mutations in an ethnic Han Chinese family from Shanxi with hereditary multiple exostoses. Polymerase chain reaction and DNA sequencing were used to screen potential mutations in EXT1 and EXT2 genes. For EXT1 gene, two synonymous mutations (P477P and E587E), three intronic mutations (c.1537 -48A>G, c.1721 +203A>G and c.1722 -103C>G) were detected. For EXT2 gene, five intronic mutations (c.-29 -148A>T, c.1080 -18T>A, c.1336 -93C>T, c.1526 -166C>T, and c.1526 -195C>T) were identified. Among these, EXT1 P477P, EXT1 E587E and EXT2 c.1080 -18T>A are polymorphisms listed by Multiple Osteochondroma Mutation Database, whilst the other 7 sites have not been reported. No mutations have been found among all exons of the EXT1 and EXT2 genes in this family. Linkage analysis is necessary for identifying the cause of this disease. Show less

Microvascular rarefaction increases vascular resistance and pressure in systemic arteries and is a hallmark of fixed essential hypertension. Preventing rarefaction by activation of angiogenic processes could lower blood pressure. Endothelial tip cells in angiogenic sprouts direct branching of microvascular networks; the process is regulated by microRNAs, particularly the miR-30 family. We investigated the contribution of miR-30 family members in arteriolar branching morphogenesis via delta-like 4 (Dll4)-Notch signaling in a zebrafish model. The miR-30 family consists of 5 members (miR-30a-e). Loss-of-function experiments showed that only miR-30a reduced growth of intersegmental arterioles involving impaired tip cell function. Overexpression of miR-30a stimulated tip cell behavior resulting in augmented branching of intersegmental arterioles. In vitro and in vivo reporter assays showed that miR-30a directly targets the Notch ligand Dll4, a key inhibitor of tip cell formation. Coadministration of a Dll4 targeting morpholino in miR-30a morphants rescued the branching defects. Conversely, conditional overexpression of Notch intracellular domain restored arteriolar branching in miR-30a gain-of-function embryos. In human endothelial cells, loss of miR-30a increased DLL4 protein levels, activated Notch signaling as indicated in Notch reporter assays, and augmented Notch downstream effector, HEY2 and EFNB2 (ephrin-B2), expression. In spheroid assays, miR-30a loss- and gain-of-function affected tip cell behavior, consistent with miR-30a targeting Dll4. Our data suggest that miR-30a stimulates arteriolar branching by downregulating endothelial Dll4 expression, thereby controlling endothelial tip cell behavior. These findings could have relevance to the rarefaction process and, therefore, to hypertension. Show less

Colonic paracellular permeability is regulated by various factors, including dynamics of the cytoskeleton. Recently, ACF7 has been found to play a critical role in cytoskeletal dynamics as an essential integrator. To elucidate the physiological importance of ACF7 and paracellular permeability, we conditionally knocked out ACF7 in the intestinal mucosa of mice. Histopathological findings indicated that ACF7 deficiency resulted in significant interstitial proliferation and columnar epithelial cell rearrangement. Decreased colonic paracellular permeability was detected using a Ussing chamber and the FITC-inulin method. In order to clarify the underlying mechanism, we further analyzed the expression levels of three important tight junction proteins. Downregulation of ZO-1, occludin and claudin-1 was identified. Immunofluorescence provided strong evidence that ZO-1, occludin and claudin-1 were weakly stained. We hypothesized that ACF7 regulates cytoskeleton dynamics to alter mucosal epithelial arrangement and colonic paracellular permeability. Show less

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α. Show less

To explore the function of PPAR γ in the goat mammary gland, we cloned the whole cDNA of the PPAR γ gene. Homology alignments revealed that the goat PPAR γ gene is conserved among goat, bovine, mouse, and human. Luciferase assays revealed that rosiglitazone enhanced the activity of the PPAR γ response element (PPRE) in goat mammary epithelial cells (GMECs). After rosiglitazone (ROSI) treatment of GMECs, there was a significant (P < 0.05) increase in the expression of genes related to triacylglycerol synthesis and secretion: LPL, FASN, ACACA, PLIN3, FABP3, PLIN2, PNPLA2, NR1H3, SREBF1, and SCD. The decreases in expression observed after knockdown of PPAR γ relative to the control group (Ad-NC) averaged 65%, 52%, 67%, 55%, 65%, 58%, 85%, 43%, 50%, and 24% for SCD, DGAT1, AGPAT6, SREBF1, ACACA, FASN, FABP3, SCAP, ATGL, and PLIN3, respectively. These results provide direct evidence that PPAR γ plays a crucial role in regulating the triacylglycerol synthesis and secretion in goat mammary cells and underscore the functional importance of PPAR γ in mammary gland tissue during lactation. Show less