High Glucose Triggers Nucleotide Imbalance through O-GlcNAcylation of Key Enzymes and Induces KRAS Mutation in Pancreatic Cells

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AICAR通过激活Foxc1信号来调节小鼠F9细胞的增殖

为了探索5-氨基咪唑-4-氨甲酰核糖核苷（AICAR）抑制小鼠F9 细胞（F9 embryonal carcinoma cells）增殖的作用机制,本文构建了Foxc1的慢病毒真核表达载体,通过实时定量PCR、免疫荧光染色、双荧光素酶报告基因检测系统以及细胞增殖检测试验,探索AICAR抑制小鼠F9细胞的增殖作用机制. 结果发现,AICAR可以在RNA和蛋白水平促进Foxc1的基因表达,并可以作用于核转录因子κB通路. 另外在培养液中添加AICAR或过表达Foxc1都能抑制F9细胞的增殖. 信号通路报告载体检测发现Foxc1可以激活核转录因子κB通路以及细胞周期相关的通路. 总之,本研究证明,AICAR 通过激活Foxc1通路及其下游多条信号通路来抑制F9细胞增殖.     

Signaling properties of 4-hydroxyalkenals formed by lipid peroxidation in diabetes

Peroxidation of polyunsaturated fatty acids is intensified in cells subjected to oxidative stress and results in the generation of various bioactive compounds, of which 4-hydroxyalkenals are prominent. During the progression of type 2 diabetes mellitus, the ensuing hyperglycemia promotes the generation of reactive oxygen species (ROS) that contribute to the development of diabetic complications. It has been suggested that ROS-induced lipid peroxidation and the resulting 4-hydroxyalkenals markedly contribute to the development and progression of these pathologies. Recent findings, however, also suggest that noncytotoxic levels of 4-hydroxyalkenals play important signaling functions in the early phase of diabetes and act as hormetic factors to induce adaptive and protective responses in cells, enabling them to function in the hyperglycemic milieu. Our studies and others′ have proposed such regulatory functions for 4-hydroxynonenal and 4-hydroxydodecadienal in insulin secreting β-cells and vascular endothelial cells, respectively. This review presents and discusses the mechanisms regulating the generation of 4-hydroxyalkenals under high glucose conditions and the molecular interactions underlying the reciprocal transition from hormetic to cytotoxic agents.     

敲低ACTL6A通过Notch1信号通路促进早幼粒细胞分化

探讨ACTL6A在人类白血病NB4细胞分化中的作用及其相关机制。我们用ATRA人为诱导NB4细胞分化,Western blotting检测ACTL6A和CD11b的表达水平变化;敲低ACTL6A,通过瑞氏染色观察NB4细胞的形态学改变,Western blotting检测ACTL6A和CD11b的表达水平变化及其相关蛋白的表达水平;敲低同时用ATRA处理NB4细胞,用流式细胞术检测分化标志物CD11b的阳性率;免疫荧光检测ACTL6A在NB4细胞中的空间定位;结果显示NB4经敲低ACTL6A后,CD11b的蛋白水平表达升高;瑞氏染色观察到分化改变;免疫荧光检测到ACTL6A主要分布于细胞核; Western blotting检测到Notch1,Hes1,Sox2蛋白表达水平明显下调。研究表明,敲低ACTL6A可以促进人类白血病NB4细胞分化;其机制涉及Notch1信号通路的抑制。     

Brain microvascular endothelium induced-annexin A1 secretion contributes to small cell lung cancer brain metastasis

Small cell lung cancer is the most aggressive histologic subtype of lung cancer, with a strong predilection for metastasizing to brain early. However, the cellular and molecular basis is poorly known. Here, we provided evidence to reveal the role of annexin A1 in small cell lung cancer metastasis to brain. Firstly, the elevated annexin A1 serum levels in small cell lung cancer patients were associated with brain metastasis. The levels of annexin A1 were also upregulated in NCI-H446 cells, a small cell lung cancer cell line, upon migration into the mice brain. More interestingly, annexin A1 was secreted by NCI-H446 cells in a time-dependent manner when co-culturing with human brain microvascular endothelial cells, which was identified with the detections of annexin A1 in the co-cultured cellular supernatants by ELISA and western blot. Further results showed that blockage of annexin A1 in the co-cultured cellular supernatants using a neutralized antibody significantly inhibited NCI-H446 cells adhesion to brain endothelium and its transendothelial migration. Conversely, the addition of Ac2-26, an annexin A1 mimic peptide, enhanced these effects. Furthermore, knockdown of annexin A1 in NCI-H446 cells prevented its transendothelial migration in vitro and metastasis to mice brain in vivo. Our data showed that small cell lung cancer cell in brain microvasculature microenvironment could express much more annexin A1 and release it outside, which facilitated small cell lung cancer cell to gain malignant properties of entry into brain. These findings provided a potential target for the management of SCLC brain metastasis.     

Efflux of a suppressive neurotransmitter, GABA, across the blood-brain barrier

In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into parietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half-life of 16.9 min. The blood-brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 x 10(-2) x min(-1)) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 microL/min/g brain) and the apparent efflux clearance (153 microL/min/g brain) indicated that the efflux clearance was at least 16-fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, beta-alanine and nipecotic acid in a concentration-dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.     

Adverse Effect of Cyclosporin A on Barrier Functions of Cerebral Microvascular Endothelial Cells After Hypoxia-reoxygenation Damage In Vitro

Hypoxia and post-hypoxic reoxygenation induces disruption of the blood–brain barrier (BBB). Alterations of the BBB function after hypoxia/reoxygenation (H/R) injury remain unclear. Cyclosporin A (CsA), a potent immunosuppressant, induces neurotoxic effects by entering the brain, although the transport of CsA across the BBB is restricted by P-glycoprotein (P-gp), a multidrug efflux pump, and tight junctions of the brain capillary endothelial cells. The aim of this study was to evaluate whether the BBB after H/R damage is vulnerable to CsA-induced BBB dysfunction. We attempted to establish a pathophysiological BBB model with immortalized mouse brain capillary endothelial (MBEC4) cells. The effects of CsA on permeability and P-gp activity of the MBEC4 cells were then examined. Exposure to hypoxia for 4 h and reoxygenation for 1 h (H/R (4 h/1 h)) produced a significant decrease in P-gp function of MBEC4 cells, without changing cell viability and permeability for sodium fluorescein and Evan’s blue-albumin at 7 days after H/R (4 h/1 h). CsA-induced hyperpermeability and P-gp dysfunction in MBEC4 monolayers at 7 days after H/R (4 h/1 h) were exacerbated. The possibility that CsA penetrates the BBB with incomplete functions in the vicinity of cerebral infarcts to induce neurotoxicity has to be considered.     

Inhibition of Transforming Growth Factor-<Emphasis Type="Bold">β</Emphasis> Production in Brain Pericytes Contributes to Cyclosporin A-Induced Dysfunction of the Blood-Brain Barrier

1. The present study was designed to clarify whether brain pericytes and pericyte-derived transforming growth factor-β1 (TGF-β1) participate in cyclosporin A (CsA)-induced dysfunction of the blood-brain barrier (BBB). 2. The presence of brain pericytes markedly aggravated CsA-increased permeability of MBEC4 cells to sodium fluorescein and accumulation of rhodamine 123 in MBEC4 cells. 3. Exposure to CsA significantly decreased the levels of TGF-β1 mRNA in brain pericytes in pericyte co-cultures. Treatment with TGF-β1 dose-dependently inhibited CsA-induced hyperpermeability and P-glycoprotein dysfunction of MBEC4 cells in pericyte co-cultures. 4. These findings suggest that an inhibition of brain pericyte-derived TGF-β1 contributes to the occurrence of CsA-induced dysfunction of the BBB.     

Permeability properties of a three-cell type in vitro model of blood-brain barrier

We previously found that RBE4.B brain capillary endothelial cells (BCECs) form a layer with blood-brain barrier (BBB) properties if co-cultured with neurons for at least one week. As astrocytes are known to modulate BBB functions, we further set a culture system that included RBE4.B BCECs, neurons and astrocytes. In order to test formation of BBB, we measured the amount of 3H-sucrose able to cross the BCEC layer in this three-cell type model of BBB. Herein we report that both neurons and astrocytes induce a decrease in the permeability of the BCEC layer to sucrose. These effects are synergic as if BCECs are cultured with both neurons and astrocytes for 5 days, permeability to sucrose decreases even more. By Western analysis, we also found that, in addition to the canonical 60 kDa occludin, anti-occludin antibodies recognize a smaller protein of 48 kDa which accumulates during rat brain development. Interestingly this latter protein is present at higher amounts in endothelial cells cultured in the presence of both astrocytes and neurons, that is in those conditions in which sucrose permeation studies indicate formation of BBB.     

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn²⁺, lipopolysaccharide, or IL-6, but the mechanism of this regulation has not been elucidated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca²⁺-uptake transporter thought to support Golgi uptake of Mn²⁺ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca²⁺ regulates ZIP8- and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an increase in cytoplasmic Ca²⁺ levels. In turn, we found increased cytoplasmic Ca²⁺ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in ⁵⁴Mn²⁺ uptake. Furthermore, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca²⁺ and ⁵⁴Mn²⁺ accumulation. While addition of Ca²⁺ positively regulated ZIP-mediated ⁵⁴Mn²⁺ uptake, we show chelation of Ca²⁺ diminished manganese transport. In conclusion, the modulation of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn²⁺ and other divalent metal ions–mediated ZIP metal transporters.     

Fibrinogen alters mouse brain endothelial cell layer integrity affecting vascular endothelial cadherin

Many inflammatory diseases are associated with elevated blood concentration of fibrinogen (Fg) leading to vascular dysfunction. We showed that pathologically high (4 mg/ml) content of Fg disrupts integrity of endothelial cell (EC) layer and causes macromolecular leakage affecting tight junction proteins. However, role of adherence junction proteins, particularly vascular endothelial cadherin (VE-cadherin) and matrix metalloproteinase-9 (MMP-9) in this process is not clear. We tested the hypothesis that at high levels Fg affects integrity of mouse brain endothelial cell (MBEC) monolayer through activation of MMP-9 and downregulation of VE-cadherin expression and in part its translocation to the cytosol.The effect of Fg on cultured MBEC layer integrity was assessed by measuring transendothelial electrical resistance. Cellular expression and translocation of VE-cadherin were assessed by Western blot and immunohistochemical analyses, (respectively). Our results suggest that high content of Fg decreased VE-cadherin expression at protein and mRNA levels. Fg induced translocation of VE-cadherin to cytosol, which led to disruption of cell-to-cell interaction and cell to subendothelial matrix attachment. Fg-induced alterations in cell layer integrity and their attachment were diminished during inhibition of MMP-9 activity.Thus Fg compromises EC layer integrity causing downregulation and translocation of VE-cadherin and through MMP-9 activation. These results suggest that increased level of Fg could play a significant role in vascular dysfunction and remodeling.     

Pathophysiological significance of cylindromatosis in the vascular endothelium and macrophages for the initiation of age-related atherogenesis

Cardiovascular disease is one of the leading causes of death in the elderly, and novel therapeutic targets against atherogenesis are urgent. The initiation of atherosclerotic changes of monocyte adhesion on the vascular endothelium and subsequent foam cell formation are noteworthy pathophysiologies when searching for strategies to prevent the progression of age-related atherosclerosis. We report the significance of the deubiquitinating enzyme cylindromatosis (CYLD) in vascular remodeling by interference with inflammatory responses regulated by NF-κB signaling. The purpose of this study was to elucidate the pathological functions of CYLD in the early phase of atherogenesis associated with aging.Treatment with inflammatory cytokines induced endogenous CYLD in aortic endothelial cells (HAECs) and THP-1?cells. siRNA-mediated CYLD silencing led to enhanced monocyte adhesion along with increased adhesion molecules in HAECs treated with TNFα. In siRNA-mediated CYLD silenced RAW 264.7 macrophages treated with oxidized LDL (oxLDL), augmented lipid accumulation was observed, along with increased expression of the class A macrophage scavenger receptor (SR-A), lectin-like oxidized LDL receptor-1 (LOX-1), CD36, fatty acid binding protein 4 (FABP4), the cholesterol ester synthase acyl-CoA cholesterol acyltransferase (ACAT1), MCP-1, and IL-1β and decreased expression of scavenger receptor class B type I (SR-BI). Intriguingly, CYLD gene expression was significantly reduced in bone marrow-derived macrophages of aged mice compared that of young mice, as well as in senescent HAECs compared with young cells.These findings suggest that age-related attenuation of CYLD expression in endothelial cells (ECs) and macrophages triggers the initiation of age-related atherogenesis by exacerbating monocyte adhesion on the endothelium and foam cell formation. CYLD in the vasculature may be a novel therapeutic target, especially in the early preventive intervention against the initiation of age-related atherogenesis.     

The importance of conserved amino acid residues in p94 protease sub-domain IIb and the IS2 region for constitutive autolysis

p94/calpain 3, a skeletal muscle-specific member of calpain protease family, is characterized by apparent Ca(2+)-independence during exhaustive autolysis and concomitant proteolysis of non-self substrates. The purpose of our study was to comprehensively profile the structural basis of p94 enabling activation in the cytosol without an extra Ca(2+). Ca(2+)-dependent p94 mutants were screened using "p94-trapping", which is an application of yeast genetic reporter system called "proteinase-trapping". Several amino acids were revealed as critical for apparent Ca(2+)-independent p94 activity. These results highlight the importance of conserved amino acids in domain IIb as well as in the p94-specific IS2 region.     

Effects of oxysterols on the blood–brain barrier: Implications for Alzheimer’s disease

Altered brain cholesterol homeostasis plays a key role in neurodegenerative diseases such as Alzheimer’s disease (AD). For a long time, the blood–brain barrier (BBB) was basically considered as a barrier isolating the brain from circulating cholesterol, however, several lines of evidence now suggest that the BBB strictly regulates the exchanges of sterol between the brain and the peripheral circulation. Oxysterols, synthesized by neurons or by peripheral cells, cross the BBB easily and modulate the expression of several enzymes, receptors and transporters which are involved not only in cholesterol metabolism but also in other brain functions. This review article deals with the way oxysterols impact BBB cells. These perspectives open new routes for designing certain therapeutical approaches that target the BBB so that the onset and/or progression of brain diseases such as AD may be modulated.     

Blood-to-brain influx transport of nicotine at the rat blood–brain barrier: Involvement of a pyrilamine-sensitive organic cation transport process

Nicotine is the most potent neural pharmacological alkaloid in tobacco, and the modulation of nicotine concentration in the brain is important for smoking cessation therapy. The purpose of this study was to elucidate the net flux of nicotine transport across the blood–brain barrier (BBB) and the major contributor to nicotine transport in the BBB. The in vivo brain-to-blood clearance was determined by a combination of the rat brain efflux index method and a rat brain slice uptake study, and the blood-to-brain transport of nicotine was evaluated by in vivo vascular injection in rats and a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13 cells) as an in vitro model of the rat BBB. The blood-to-brain nicotine influx clearance was obtained by integration plot analysis as 272 μL/(min g brain), and this value was twofold greater than the brain-to-blood efflux clearance (137 μL/(min g brain)). Thus, it is suggested that the net flux of nicotine transport across the BBB is dominated by blood-to-brain influx transport. In vivo blood-to-brain nicotine transport was inhibited by pyrilamine. [³H]Nicotine uptake by TR-BBB13 cells exhibited time-, temperature-, and concentration-dependence with a K_m value of 92 μM. Pyrilamine competitively inhibited nicotine uptake by TR-BBB13 cells with a K_i value of 15 μM, whereas substrates and inhibitors of organic cation transporters had little effect. These results suggest that pyrilamine-sensitive organic cation transport process(es) mediate blood-to-brain influx transport of nicotine at the BBB, and this is expected to play an important role in regulating nicotine-induced neural responses.     

Recent progresses in gene delivery-based bone tissue engineering

Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches.     

The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis : Application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus

The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N = 13) and infected with CAEV (N = 13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240 days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and – according to the geNorm results – the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80 °C was also proved.