[Progress in caveolae and its proteomic research].
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
Yi Zhu
Publications and source records attributed to Yi Zhu.
Characterization of (2R, 3S)-2-([[4-(2-butynyloxy)phenyl]sulfonyl]amino)-N,3-dihydroxybutanamide, a potent and selective inhibitor of TNF-alpha converting enzyme.
TNF-alpha converting enzyme (TACE) is a validated therapeutic target for the development of oral tumor necrosis factor-alpha (TNF-alpha) inhibitors. Here we report the pre-clinical results and characterization of a selective and potent TACE inhibitor, (2R, 3S)-2-([[4-(2-butynyloxy)phenyl]sulfonyl]amino)-N,3-dihydroxybutanamide (TMI-2), in various in vitro and in vivo assays. TMI-2 is a potent TACE inhibitor in an enzymatic FRET assay (IC50=2 nM). It is more than 250-fold selective over MMP-1, -7, -9, -14, and ADAM-10 in vitro. In cell-based assays and human whole blood, TMI-2 inhibits lipopolysaccharide (LPS)-induced TNF secretion with IC50s<1 uM. Importantly, TMI-2 inhibits the spontaneous release of TNF-alpha in human synovium tissue explants of rheumatoid arthritis patients with an IC50 of 0.8 microM. In vivo, TMI-2 potently inhibits LPS-induced TNF-alpha production in mice (ED50=3 mg/kg). In the adjuvant-induced arthritis (AIA) model in rats, treatment with TMI-2 at 30 mg/kg and 100 mg/kg p.o. b.i.d. was highly effective in reducing joint arthritis scores. In a semi-therapeutic collagen-induced arthritis (CIA) model in mice, TMI-2 is highly effective in reducing disease severity scores after oral treatment at 100 mg/kg twice per day. In summary, TMI-2 is a potent and selective TACE inhibitor that inhibits TNF-alpha production and reduces the arthritis scores in pre-clinical models. TMI-2 represents a novel class of TACE inhibitors that may be effective and beneficial in the treatment of rheumatoid arthritis as well as other TNF-mediated inflammatory autoimmune diseases.
ADAM Proteins↗
Intermittent high altitude hypoxia protects the heart against lethal Ca2+ overload injury.
Adaptation to intermittent high altitude (IHA) hypoxia can protect the heart against ischemia-reperfusion injury. In view of the fact that both Ca2+ paradox and ischemia-reperfusion injury are associated with the intracellular Ca2+ overload, we tested the hypothesis that IHA hypoxia may protect hearts against Ca2+ paradox-induced lethal injury if its cardioprotection bases on preventing the development of intracellular Ca2+ overload. Langendorff-perfused hearts from normoxic and IHA hypoxic rats were subjected to Ca2+ paradox (5 min of Ca2+ depletion followed by 30 min of Ca2+ repletion) and the functional, biochemical and pathological changes were investigated. The Ca2+ paradox incapacitated the contractility of the normoxic hearts, whereas the IHA hypoxic hearts significantly preserved contractile activity. Furthermore, the normoxic hearts subjected to Ca2+ paradox exhibited a marked reduction in coronary flow, increase in lactate dehydrogenase release, and severe myocyte damage. In contrast, these changes were significantly prevented in IHA hypoxic hearts. We, then, tested and confirmed our hypothesis that the protective mechanisms are mediated by mitochondria ATP-sensitive potassium channels (mitoKATP) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), as the protective effect of IHA hypoxia was abolished by 5-hydroxydecanoate, a selective mitoKATP blocker, and significantly attenuated by KN-93, a CaMKII inhibitor. In conclusion, our studies offer for the first time that IHA hypoxia confers cardioprotection against the lethal injury of Ca2+ paradox and give biochemical evidence for the protective mechanism of IHA hypoxia. We propose that researches in this area may lead a preventive regimen against myocardial injury associated with Ca2+ overload.
Adaptation, Physiological↗
Drosophila homeodomain protein Nkx6 coordinates motoneuron subtype identity and axonogenesis.
The regulatory networks acting in individual neurons to control their stereotyped differentiation, connectivity, and function are not well understood. Here, we demonstrate that homeodomain protein Nkx6 is a key member of the genetic network of transcription factors that specifies neuronal fates in Drosophila. Nkx6 collaborates with the homeodomain protein Hb9 to specify ventrally projecting motoneuron fate and to repress dorsally projecting motoneuron fate. While Nkx6 acts in parallel with hb9 to regulate motoneuron fate, we find that Nkx6 plays a distinct role to promote axonogenesis, as axon growth of Nkx6-positive motoneurons is severely compromised in Nkx6 mutant embryos. Furthermore, Nkx6 is necessary for the expression of the neural adhesion molecule Fasciclin III in Nkx6-positive motoneurons. Thus, this work demonstrates that Nkx6 acts in a specific neuronal population to link neuronal subtype identity to neuronal morphology and connectivity.
Animals↗
Sterol-responsive element-binding protein (SREBP) 2 down-regulates ATP-binding cassette transporter A1 in vascular endothelial cells: a novel role of SREBP in regulating cholesterol metabolism.
ATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of cholesterol efflux from cells to apolipoproteins, whereas sterol-responsive element-binding protein 2 (SREBP2) is the key protein regulating cholesterol synthesis and uptake. We investigated the regulation of ABCA1 by SREBP2 in vascular endothelial cells (ECs). Our results showed that sterol depletion activated SREBP2 and increased its target, low density lipoprotein receptor mRNA, with a concurrent decrease in the ABCA1 mRNA. Transient transfection analysis revealed that sterol depletion decreased the ABCA1 promoter activity by 50%, but low density lipoprotein receptor promoter- and the sterol-responsive element-driven luciferase activities were increased. Overexpression of the N terminus of SREBP2 (SREBP2(N)), an active form of SREBP2, also inhibited the ABCA1 promoter activity. Functionally adenovirus-mediated SREBP2(N) expression increased cholesterol accumulation and decreased apoA-I-mediated cholesterol efflux. The conserved E-box motif was responsible for the SREBP2(N)-mediated inhibition since mutation of the E-box increased the basal activity of the ABCA1 promoter and abolished the inhibitory effect of SREBP2(N). Furthermore sterol depletion and SREBP2(N) overexpression induced the binding of SREBP2(N) to both consensus and ABCA1-specific E-box. Chromatin immunoprecipitation assay demonstrated that serum starvation enhanced the association of SREBP2 and the ABCA1 promoter in ECs. To correlate this mechanism pathophysiologically, we found that oscillatory flow caused the activation of SREBP2 and therefore attenuated ABCA1 promoter activity in ECs. Thus, this SREBP-regulated mechanism may control the efflux of cholesterol, which is a newly defined function of SREBP2 in ECs in addition to its role in cholesterol uptake and biosynthesis.
ATP Binding Cassette Transporter 1↗
Simvastatin induces heme oxygenase-1: a novel mechanism of vessel protection.
BACKGROUND: Evidence from experimental and clinical studies indicates that statins can protect the vessel wall through cholesterol-independent mechanisms. The "pleiotropic" effects include the prevention of inflammation and proliferation of vascular cells. Here, we studied whether heme oxygenase-1 (HO-1), an important cytoprotective molecule, is induced by simvastatin and the role of HO-1 in the pleiotropic effects of simvastatin. METHODS AND RESULTS: Human and rat aortic smooth muscle cells treated with simvastatin showed an elevated level of HO-1 for up to 24 hours. The induction of HO-1 by simvastatin was not found in cultured endothelial cells and macrophages. Injecting C57BL/6J mice intraperitoneally with simvastatin increased the level of HO-1 in vascular SMCs (VSMCs) in the tunica media. Treating VSMCs with zinc protoporphyrin, an HO-1 inhibitor, or HO-1 small interfering RNA (siRNA) blocked the antiinflammatory effect of simvastatin, including the inhibition of nuclear factor-kappaB activation and nitric oxide production. Blockade of HO-1 also abolished the simvastatin-induced p21(Waf1) and the associated antiproliferative effect. Simvastatin activated p38 and Akt in VSMCs, and the respective inhibitors of p38 and phosphoinositide 3-kinase (PI3K) greatly reduced the level of simvastatin-induced HO-1, which suggests the involvement of p38 and the PI3K-Akt pathway in HO-1 induction. CONCLUSIONS: Simvastatin activates HO-1 in VSMCs in vitro and in vivo. The antiinflammatory and antiproliferative effects of simvastatin occur largely through the induced HO-1.
Animals↗
Laminar flow activates peroxisome proliferator-activated receptor-gamma in vascular endothelial cells.
BACKGROUND: Steady laminar flow is atheroprotective, in part because of its antiinflammatory effects on vascular endothelial cells (ECs). We studied the activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) in ECs in response to laminar flow and the associated antiinflammatory effect. METHODS AND RESULTS: Using flow channel with cultured ECs, we found that laminar flow activated the PPARgamma-mediated PPAR-responsive element (PPRE) activity and increased the mRNA encoding CD36, a PPARgamma-targeted gene. Analysis of the CD36 promoter revealed that PPRE was required for flow activation. Laminar flow induced the GAL-PPARgamma-LBD fusion protein, which suggests that flow activation of PPARgamma was ligand dependent. The pharmaceutical inhibitors of phospholipase A2 (PLA2) and cytochrome P450 epoxygenases (CYP450s) were able to block the laminar flow-activated PPARgamma. We also showed that lipid extracts from flow media contained ligands for the activation of PPARgamma in other cell types. This paracrine activation exerted antiinflammatory effects in ECs and THP-1 cells, including the suppression of cytokine-induced nuclear factor-kappaB activation and expression of intercellular adhesion molecule-1. CONCLUSIONS: Laminar flow activates endogenous PPARgamma in ECs, which is ligand dependent. The flow production of PPARgamma ligands is through the PLA2-CYP450 pathway, and the induced PPARgamma ligands exert antiinflammatory effects in several types of cells.
Animals↗
Liver X receptor-alpha mediates cholesterol efflux in glomerular mesangial cells.
Lipid-mediated injury plays an important role in the pathogenesis of many renal diseases including diabetic nephropathy. Liver X receptor-alpha (LXRalpha) is an intracellular sterol sensor that regulates expression of genes controlling cholesterol absorption, excretion, catabolism, and cellular efflux. The present study was aimed at examining the role of LXRalpha in cholesterol metabolism in glomerular mesangial cells. A 1,561-bp fragment of full-length rabbit LXR cDNA was cloned. The deduced protein sequence exhibited 92.4 and 89.2% identity to human and mouse LXRalpha, respectively. Tissue distribution studies showed that rabbit LXRalpha was expressed in the liver, spleen, and kidney. In situ hybridization and RT-PCR assays further indicated that LXRalpha mRNA was widely expressed in the kidney and present in every nephron segment including the glomeruli. To determine intrarenal regulation of LXRalpha, rabbits were treated with thiazolidinedione (TZD) peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists, which have been previously shown to enhance LXRalpha expression via PPARgamma and increase cholesterol efflux in macrophages. The results showed that glomerular LXRalpha expression was markedly induced by TZDs. In cultured rabbit mesangial cells, LXRalpha mRNA and protein were detected by RT-PCR and immunoblotting. Treatment of mesangial cells with a specific LXRalpha agonist, TO-901317, significantly increased basal and apolipoprotein AI-mediated cholesterol efflux and markedly enhanced the promoter activity of an LXRalpha target gene, ATP-binding cassette transporter A1 (ABCA1). In conclusion, LXRalpha is expressed in renal glomeruli and functionally present in mesangial cells where its activation mediates cholesterol efflux via ABCA1. These data suggest that LXRalpha may be a potential therapeutic target for treating lipid-related renal glomerular disease.
3' Flanking Region↗
Vascular endothelial growth factor activation of sterol regulatory element binding protein: a potential role in angiogenesis.
By stimulating the migration and proliferation of endothelial cells (ECs), vascular endothelial growth factor (VEGF) is a potent angiogenic factor. However, the molecular mechanism involved in the VEGF-induced angiogenesis remains elusive. We hypothesized that sterol regulatory element binding proteins (SREBPs), transcription factors governing cellular lipid homeostasis, play an important role in regulating angiogenesis in response to VEGF. VEGF activated SREBP1 and SREBP2 in ECs, as demonstrated by the increased SREBPs, their cleavage products, and the upregulation of the targeted genes. VEGF-induced SREBP activation depended on SREBP cleavage-activating protein (SCAP), because knocking down SCAP by RNA interference (RNAi) inhibited SREBP activation in response to VEGF. SREBP activation was also blocked by 25-hydroxycholesterol (25-HC). To verify the functional implication of SREBPs in VEGF-induced angiogenesis, we tested the role of SREBPs in EC migration and proliferation. SCAP RNAi or 25-HC inhibited VEGF-induced pseudopodia extension and migration of ECs. Both treatments inhibited VEGF-induced EC proliferation, with cell growth arrested at the G(0)/G(1) phase and a concomitant decrease of the S phase. Blocking the PI3K-Akt pathway inhibited the VEGF-activated SREBPs, demonstrating that PI3K-Akt regulates SREBPs. Consistent with our in vitro data, SREBP1 was detected in newly developed microvasculatures in a rabbit skin partial-thickness wound-healing model. SREBP inhibition also markedly suppressed VEGF-induced angiogenesis in chick embryos. In summary, this study identifies SREBPs as the key molecules in regulating angiogenesis in response to VEGF.
Animals↗
Mechanisms of acupuncture and moxibustion in regulation of epithelial cell apoptosis in rat ulcerative colitis.
AIM: To investigate the effect of acupuncture and moxibustion on epithelial cell apoptosis and expression of Bcl-2, Bax, fas and FasL proteins in rat ulcerative colitis. METHODS: A rat model of ulcerative colitis was established by immunological methods and local stimulation. All rats were randomly divided into model control group (MC), electro-acupuncture group (EA), herbs-partition moxibustion group (HPM). Normal rats were used as normal control group (NC). Epithelial cell apoptosis and expression of Bcl-2, Bax, fas and FasL proteins were detected by TUNEL and immunohistochemical method respectively. RESULTS: The number of epithelial cell apoptosis in MC was significantly higher than that in NC, and was markedly decreased after the treatment with herbs-partition moxibustion or electro-acupuncture. The expression of Bcl-2, Bax, fas and FasL in colonic epithelial cells in MC was higher than that in NC, and was markedly down- regulated by herbs-partition moxibustion or electro-acupuncture treatment. CONCLUSION: The pathogenesis of ulcerative colitis in rats involves abnormality of apoptosis. Acupuncture and moxibustion can regulate the expression of Bcl-2, Bax, fas and FasL proteins and inhibit the apoptosis of epithelial cells of ulcerative colitis in rats by Bcl-2/Bax, fas/FasL pathways.
Acupuncture↗
ATF6 modulates SREBP2-mediated lipogenesis.
Activating transcription factor 6 (ATF6) and sterol regulatory element-binding proteins (SREBPs) are activated by proteolytic cleavage. The ensuing nuclear translocation of their N-termini (i.e., ATF6(N) and SREBP(N)) activates the respective target genes involved in unfolded protein response and lipogenesis. Here, we report that glucose deprivation activated ATF6 but suppressed the SREBP2-regulated transcription. Overexpression of ATF6(N) had similar inhibitory effects on SREBP2-targeted genes. The blockade of ATF6 cleavage by BiP/grp78 reversed this inhibitory effect. GST pull-down and immunoprecipitation assays revealed that ATF6(N) bound to SREBP2(N). Deletion analysis of the various functional domains of ATF6 indicated that the interaction was through its leucine-zipper domain. Chromatin immunoprecipitation assays revealed that ATF6(N) formed a complex with the SRE-bound SREBP2(N). The attenuated transcriptional activity of SREBP2 was due, in part, to the recruitment of HDAC1 to the ATF6-SREBP2 complex. As a functional consequence, the lipogenic effect of SREBP2(N) in liver cells was suppressed by ATF6(N). Our results provide a novel mechanism by which ATF6 antagonizes SREBP2 to regulate the homeostasis of lipid and glucose.
Activating Transcription Factor 6↗
Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations.
Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains.
Chromatography, High Pressure Liquid↗
Atomic force microscopy studies on DNA structural changes induced by vincristine sulfate and aspirin.
We report that atomic force microscopy (AFM) studies on structural variations of a linear plasmid DNA interact with various concentrations of vincristine sulfate and aspirin. The different binding images show that vincrinstine sulfate binding DNA chains caused some loops and cleavages of the DNA fragments, whereas aspirin interaction caused the width changes and conformational transition of the DNA fragments. Two different DNA structural alternations could be explained by the different mechanisms of the interactions with these two components. Our work indicates that the AFM is a powerful tool in studying the interaction between DNA and small molecules.
Aspirin↗
Mutational analysis of the signal-sensing domain of ResE histidine kinase from Bacillus subtilis.
The Bacillus subtilis ResD-ResE two-component regulatory system activates genes involved in nitrate respiration in response to oxygen limitation or nitric oxide (NO). The sensor kinase ResE activates the response regulator ResD through phosphorylation, which then binds to the regulatory region of genes involved in anaerobiosis to activate their transcription. ResE is composed of an N-terminal signal input domain and a C-terminal catalytic domain. The N-terminal domain contains two transmembrane subdomains and a large extracytoplasmic loop. It also has a cytoplasmic PAS subdomain between the HAMP linker and C-terminal kinase domain. In an attempt to identify the signal-sensing subdomain of ResE, a series of deletions and amino acid substitutions were generated in the N-terminal domain. The results indicated that cytoplasmic ResE lacking the transmembrane segments and the extracytoplasmic loop retains the ability to sense oxygen limitation and NO, which leads to transcriptional activation of ResDE-dependent genes. This activity was eliminated by the deletion of the PAS subdomain, demonstrating that the PAS subdomain participates in signal reception. The study also raised the possibility that the extracytoplasmic region may serve as a second signal-sensing subdomain. This suggests that the extracytoplasmic region could contribute to amplification of ResE activity leading to the robust activation of genes required for anaerobic metabolism in B. subtilis.
Amino Acid Substitution↗
[Misdiagnosis of eyelid myoclonia as Tic's disorder: a case report].
Explore the source record for details and available documents.
Diagnostic Errors↗
Low density lipoprotein induces eNOS translocation to membrane caveolae: the role of RhoA activation and stress fiber formation.
A decrease in the bioavailability of endothelium-derived nitric oxide (NO) is linked to hypercholesterolemia. However, the mechanism by which low density lipoprotein (LDL) mediates endothelial NO synthase (eNOS) dysfunction remains controversial. We investigate the effect of LDL on eNOS regulation in human endothelial cells (ECs). In cultured ECs, a high level of LDL increased the abundance of eNOS and caveolin-1 (Cav-1) in the membrane caveolae and the association of eNOS with Cav-1. Furthermore, it decreased the basal level of NO and blocked NO production stimulated by the calcium ionophore A23187. LDL exposure also increased the formation of stress fibers and the membrane translocation of eNOS. These effects can be blocked by cytochalasin D, an actin cytoskeleton disruptor. In revealing the mechanism underlying the translocation of eNOS, we found that a high level of LDL increased the level of membrane-associated and GTP-formed RhoA and activated the RhoA downstream kinase ROCK-1 activity. Y-27632, a specific inhibitor of ROCK-1, blocked LDL-induced stress fiber formation, eNOS translocation and NO production. In conclusion, a high level of LDL increases the movement of eNOS to membrane caveolae via the increased stress fibers. The RhoA-mediated pathway may play a crucial role in this process in vascular ECs.
Amides↗
Clinical course of patients with WASP gene mutations.
Mutations of the Wiskott-Aldrich syndrome protein (WASP) gene result either in the classic Wiskott-Aldrich syndrome (WAS) or in a less severe form, X-linked thrombocytopenia (XLT). A phenotype-genotype correlation has been reported by some but not by other investigators. In this study, we characterized WASP gene mutations in 50 Japanese patients and analyzed the clinical phenotype and course of each. All patients with missense mutations were WASP-positive. In contrast, patients with nonsense mutations, large deletions, small deletions, and small insertions were WASP-negative. Patients with splice anomalies were either WASP-positive or WASP-negative. The clinical phenotype of each patient was correlated with the presence or absence of WASP. Lack of WASP expression was associated with susceptibility to bacterial, viral, fungal, and Pneumocystis carinii infections and with severe eczema, intestinal hemorrhage, death from intracranial bleeding, and malignancies. Rates for overall survival and survival without intracranial hemorrhage or other serious complications were significantly lower in WASP-negative patients. This analysis provides evidence for a strong phenotype-genotype correlation and demonstrates that WAS protein expression is a useful tool for predicting long-term prognosis for patients with WAS/XLT. Based on data presented here, hematopoietic stem cell transplantation should be considered, especially for WASP-negative patients, while the patients are young to improve prognosis.
Adolescent↗
Sex- and age-dependent human transcriptome variability: implications for chronic heart failure.
Heart failure (HF) is the end result of progressive and diverse biological adaptations within the diseased myocardium. We used cDNA microarrays and quantitative PCR to examine the transcriptomes of 38 left ventricles from failing and nonfailing human myocardium. After identification of a pool of putative HF-responsive candidate genes by microarrays on seven nonfailing and eight failing hearts, we used quantitative PCR and a general linear statistical model in a larger sample set (n = 34) to validate and examine the role of contributing biological variables (age and sex). We find that most HF-candidate genes (transcription factors, Cebpb, Npat; signaling molecules, Map2k3, Map4k5; extracellular matrix proteins, Lum, Cola1; and metabolic enzymes, Mars) demonstrated significant changes in gene expression; however, the majority of differences among samples depended on variables such as sex and age, and not on HF alone. Some HF-responsive gene products also demonstrated highly significant changes in expression as a function of age and/or sex, but independent of HF (Ngp1, Cd163, and Npat). These results emphasize the need to account for biological variables (HF, sex and age interactions) to elucidate genomic correlates that trigger molecular pathways responsible for the progression of HF syndromes.
Adult↗