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101.
Kuhlmann CR Gerigk M Bender B Closhen D Lessmann V Luhmann HJ 《Life sciences》2008,82(25-26):1281-1287
Glutamate is an important excitatory amino acid in the central nervous system. Under pathological conditions glutamate levels dramatically increase. Aim of the present study was to examine whether the HMG-CoA inhibitor fluvastatin prevents glutamate-induced blood-brain-barrier (BBB) disruption. Measurements of transendothelial electrical resistance (TEER) were performed to analyze BBB integrity in an in vitro co-culture model of brain endothelial and glial cells. Myosin light chain (MLC) phosphorylation was detected by immunohistochemistry, or using the in-cell western technique. Intracellular Ca2+ and reactive oxygen species (ROS) levels were analyzed using the fluorescence dyes Ca-green or DCF. Glutamate induced a time- (1-3 h) and concentration- (0.25-1 mmol/l) dependent decrease of TEER values that was blocked by the NMDA-receptor antagonist MK801, the Ca2+ chelator BAPTA, the NAD(P)H-oxidase inhibitor apocynin and the MLC-kinase inhibitor ML-7. Furthermore we observed a concentration-dependent increase of intracellular Ca2+ and ROS after glutamate application. Glutamate caused an increase of MLC phosphorylation that was antagonized by apocynin, or BAPTA, indicating that Ca2+ and ROS signaling is involved in the activation of the contractile machinery. Fluvastatin (10-25 micromol/l) completely abolished the glutamate-induced barrier disruption and oxidative stress. The BBB-protecting effect of fluvastatin was completely lost if the cells were treated with the nitric oxide (NO) synthase inhibitor L-NMMA (300 micromol/l). In the present study we demonstrated that glutamate-induced BBB disruption involves Ca2+ signalling via NMDA receptors, which is followed by an increased ROS generation by the NAD(P)H-oxidase. This oxidative stress then activates the MLC kinase. Fluvastatin preserves barrier function in a NO-dependent way and reduces glutamate-induced oxidative stress. 相似文献
102.
Polysialic acid regulates the clustering, migration, and neuronal differentiation of progenitor cells in the adult hippocampus 总被引:1,自引:0,他引:1
Burgess A Wainwright SR Shihabuddin LS Rutishauser U Seki T Aubert I 《Developmental neurobiology》2008,68(14):1580-1590
Newborn cells of the adult dentate gyrus in the hippocampus are characterized by their abundant expression of polysialic acid (PSA), a carbohydrate attached to the neural cell adhesion molecule (NCAM). PSA+ newborn cells of the dentate gyrus form clusters with proliferating neural progenitor cells, migrate away from these clusters, and terminally differentiate. To identify the roles of PSA in the development of adult progenitors of the dentate gyrus, we injected endoneuraminidase N (endoN) into the hippocampus of adult rats to specifically cleave PSA from NCAM. Two days later, we administered the mitotic marker, 5-bromo-2'-deoxyuridine (BrdU). Three days after BrdU injection, BrdU+ cells were found inside and outside the clusters of newborn cells. In endoN-treated animals, the total number of BrdU+ cells was not changed but significantly more BrdU+ cells were present within clusters, suggesting that PSA normally facilitates the migration of progenitors away from the clusters. Seven days post-BrdU injection, endoN-treated animals had significantly more BrdU+ cells which were also positive for the mature neuronal nuclear marker NeuN compared with controls, indicating that the loss of PSA from progenitor cells increases neuronal differentiation. This report is the first demonstration that PSA is involved in controlling the spatio-temporal neuronal maturation of adult hippocampal progenitors in the normal brain. In vitro, the removal of PSA from adult-derived neural progenitors significantly enhanced neuronal differentiation, strengthening our in vivo findings and indicating that PSA removal on isolated progenitor cells, apart from a complex in vivo environment, induces neuronal maturation. 相似文献
103.
Moshal KS Zeldin DC Sithu SD Sen U Tyagi N Kumar M Hughes WM Metreveli N Rosenberger DS Singh M Vacek TP Rodriguez WE Ayotunde A Tyagi SC 《Journal of cellular physiology》2008,215(3):771-781
Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase-9 (MMP-9). Cytochrome P450 (CYP) epoxygenase-2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF-kappabeta. However, the underlying molecular mechanisms for MMP-9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)-induced MMP-9 activation. CYP2J2 was over-expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF-kappabeta-mediated MMP-9 regulation were evaluated using Western blot, in-gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K-dependent AKT signal. Hcy induced the nuclear translocation of NF-kappabeta via downregulation of IKbetaalpha (endogenous cytoplasmic inhibitor of NF-kappabeta). Hcy induced MMP-9 activation by increasing NF-kappabeta-DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9-EET phosphorylated the AKT and attenuated Hcy-induced MMP-9 activation. This occurred, in part, by the inhibition of NF-kappabeta nuclear translocation, NF-kappabeta-DNA binding and activation of IKbetaalpha. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP-9 signal. 相似文献
104.
Tamgüney T Zhang C Fiedler D Shokat K Stokoe D 《Experimental cell research》2008,314(11-12):2299-2312
3-phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several kinases in the cAMP-dependent, cGMP-dependent and protein kinase C (AGC) family. Many putative PDK1 substrates have been identified, but have not been analyzed following transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the ATP binding pocket of wild type (WT) kinases. When expressed in PDK1(-/-) ES cells, PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 optimally inhibited the phosphorylation of PDK1 targets in PDK1(-/-) ES cells expressing PDK1 L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, but revealed distinct dephosphorylation kinetics. While PDK1 inhibition had little effect on cell growth, it sensitized cells to apoptotic stimuli. Furthermore, PDK1 loss abolished growth of allograft tumors. Taken together we describe a model system that allows for acute and reversible inhibition of PDK1 in cells, to probe biochemical and biological consequences. 相似文献
105.
Circulating blood cell lipid composition may become increasingly important to provide new insights into cellular lipid abnormalities in diseases. Here we compared lipid species in monocytes, lymphocytes, granulocytes, platelets and red blood cells (RBC) of healthy volunteers using electrospray ionization tandem mass spectrometry and detected striking differences among the examined blood cells. The different cell types were characterized by unique lipid class and lipid species pattern. The predominant lipid classes were phosphatidylcholine (PC) and free cholesterol (FC) with cell type specific PC/FC ratios as markers of membrane fluidity which was 1.9 in monocytes, 1.3 in lymphocytes, 1.1 in granulocytes, 0.8 in platelets and 0.3 in RBC, respectively. Beside a three-fold elevated ceramide level of 2.6 mol%, granulocytes revealed the highest percentage of phosphatidylethanolamine-based plasmalogens and a decreased fraction of highly polyunsaturated (> or =3 double bonds) species compared to other cell types. Furthermore RBC showed a remarkable shift of glycerophospholipid chain length and platelets a nearly 4-fold increase of the cholesterol ester (CE) 18:2 (linoleic acid) fraction (55 mol% of total CE). In conclusion, the current study is a detailed comparison of lipid species in circulating blood cells of healthy human donors. This work could be a reference for studies in different patient cohorts directed towards discovery of novel lipid biomarkers in circulating blood cells. 相似文献
106.
107.
YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids 总被引:2,自引:0,他引:2
Konstanze Beck Gottfried Eisner Dorothea Trescher Ross E. Dalbey Josef Brunner Matthias Müller 《EMBO reports》2001,2(8):709-714
Like its mitochondrial homolog Oxa1p, the inner membrane protein YidC of Escherichia coli is involved in the integration of membrane proteins. We have analyzed individual insertion steps of the polytopic E. coli membrane protein MtlA targeted as ribosome-nascent chain complexes to inner membrane vesicles. YidC can accommodate at least the first two transmembrane segments of MtlA at the protein lipid interface and retain them even though the length of the nascent chain would amply allow insertion into membrane lipids. An even longer insertion intermediate of MtlA is described that still has the first transmembrane helix bound to YidC while the third contacts SecE and YidC during integration. Our findings suggest that YidC forms a contiguous integration unit with the SecYE translocon and functions as an assembly site for polytopic membrane proteins mediating the formation of helix bundles prior to their release into the membrane lipids. 相似文献
108.
109.
Laura Coombes Rob Tierney Peter Rigsby Dorothea Sesardic Paul Stickings 《Biologicals》2012,40(6):466-472
Consistency of production is recognised as an important aspect of vaccine manufacture and suitably validated in vitro assays are required for quality control testing of these products. For the manufacture and batch release of tetanus vaccines, antigen content and integrity, and degree of adsorption of antigen to the adjuvant are critical parameters that should be monitored for consistency. Here we describe the development and use of an Enzyme Linked Immunosorbent Assay (ELISA) to quantify tetanus antigen in combined vaccine products and to measure the degree of adsorption of antigen to adjuvant. Whilst the antigen assay cannot be assumed to predict potency for different products, it can be used as part of a panel of in vitro methods to provide a more informative product profile and to monitor trends in production. The antigen assay is particularly valuable for providing quantitative information on every final lot when modifications of in vivo potency tests, such as single dilution assays, are used. 相似文献
110.
Human jaw periosteum tissue contains osteoprogenitors that have potential for tissue engineering applications in oral and maxillofacial surgeries. To isolate osteoprogenitor cells from heterogeneous cell populations, we used the specific mesenchymal stem cell antigen-1 (MSCA-1) antibody and compared two magnetic separation methods. We analyzed the obtained MSCA-1+ and MSCA-1− fractions in terms of purity, yield of positive/negative cells and proliferative and mineralization potentials. The analysis of cell viability after separation revealed that the EasySep method yielded higher viability rates, whereas the flow cytometry results showed a higher purity for the MACS-separated cell fractions. The mineralization capacity of the osteogenic induced MSCA-1+ cells compared with the MSCA-1− controls using MACS was 5-fold higher, whereas the same comparison after EasySep showed no significant differences between both fractions. By analyzing cell proliferation, we detected a significant difference between the proliferative potential of the osteogenic cells versus untreated cells after the MACS and EasySep separations. The differentiated cells after MACS separation adjusted their proliferative capacity, whereas the EasySep-separated cells failed to do so. The protein expression analysis showed small differences between the two separation methods. Our findings suggest that MACS is a more suitable separation method to isolate osteoprogenitors from the entire jaw periosteal cell population. 相似文献