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Berlin Brandenburg

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  • 1
    Language: English
    In: Physiological genomics, 21 November 2011, Vol.43(22), pp.1255-62
    Description: Maternal lipoproteins have been studied extensively in human pregnancies, but little is known about the role of fetal lipoproteins. The vascularized human placenta interfaces between the mother and fetus to transfer nutrients for sustaining pregnancy. Unlike that of adults, fetal high-density lipoprotein (HDL), which is in contact with placental vessels, is characterized by a high proportion of apolipoprotein E (apoE). We hypothesize this unique composition of fetal HDL affects key functions of the growing fetal tissues. The aim was to identify genes regulated by apoE-HDL by incubating human placental endothelial cells (HPEC) with either fetal HDL or apoE-rich reconstituted HDL particles (apoE-rHDL). HPEC were exposed to 15 μg/ml fetal HDL, 15 μg/ml apoE-rHDL, or medium for 16 h, respectively. Microarray analysis determined genes regulated by fetal HDL and apoE. Characterization of HDL particles revealed a different hydrodynamic radius for apoE-rHDL (13.70 nm) compared with fetal HDL (18.11 nm). Stepwise gene clustering after microarray experiments identified 79 differentially expressed genes (P 〈 0.05) when cells were exposed to HDL compared with controls. Among them 16 genes were downregulated, whereas five genes were upregulated by twofold, respectively. When HPEC were incubated with apoE-rHDL 18-fold more genes (1,417, 12% of transcripts) were regulated (P 〈 0.05) in contrast to HDL. Thereof, 172 genes were downregulated and 376 genes upregulated (twofold). In the common subset of 38 genes regulated by both HDL particles, genes involved in cholesterol biosynthesis and cell protection prevailed. Strikingly, results suggest that HDL has the capability of regulating metallothioneins, which may have an effect on oxidative stress in HPEC.
    Keywords: Gene Expression Regulation ; Apolipoproteins E -- Genetics ; Endothelial Cells -- Metabolism ; Lipoproteins, HDL -- Genetics ; Placenta -- Metabolism
    ISSN: 10948341
    E-ISSN: 1531-2267
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  • 2
    Language: English
    In: Cellular Physiology and Biochemistry, June 2012, Vol.29(5-6), pp.809-818
    Description: Background: Cardiac action potential repolarisation is determined by K+ currents including IKs. IKs channels are heteromeric channels composed of KCNQ1 and KCNE E-subunits. Mutations in KCNQ1 are associated with sinus bradycardia, familial atrial fibrillation (fAF) and/or short QT syndrome as a result of gain-of-function, and long QT syndrome (LQTS) due to loss-of-function in the ventricles. Here, we report that the missense mutation R231C located in S4 voltage sensor domain is associated with a combined clinical phenotype of sinus bradycardia, fAF and LQTS. We aim to understand the molecular basis of the complex clinical phenotype. Methods: We expressed and functionally analyzed the respective channels kinetics in Xenopus laevis oocytes. The molecular nature of the residue R231 was studied by homology modeling and molecular dynamics simulation. Results: As a result, the mutation reduced voltage sensitivity of channels, possibly due to neutralization of the positive charge of the arginine side chain substituted by cysteine. Modeling suggested that the charge carrying side chain of R231 is positioned suitably to transfer transmembrane voltages into conformational energy. Further, the mutation altered the functional interactions with KCNE subunits. Conclusion: The mutation acted in a E-subunit dependent manner, suggesting IKs function altered by the presence of different KCNE subunits in sinus node, atria and ventricles as the molecular basis of sinus bradycardia, fAF and LQTS in mutation carriers.
    Keywords: Original Paper ; Kcne ; Heart ; Arrhythmia ; Oocyte ; Kv7.1 ; Kvlqt1 ; Biology ; Chemistry
    ISSN: 1015-8987
    E-ISSN: 1421-9778
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  • 3
    Language: English
    In: Journal of Biological Chemistry, 08/29/1997, Vol.272(35), pp.22173-22181
    Description: In the present study, we show that Fas receptor ligation or cellular treatment with synthetic C sub(6)-ceramide results in activation or phosphorylation, respectively, of the small G-protein Rac1, Jun N-terminal kinase (JNK)/p38 kinases (p38-K), and the transcription factor GADD153. A signaling cascade from the Fas receptor via ceramide, Ras, Rac1, and JNK/p38-K to GADD153 is demonstrated employing transfection of transdominant inhibitory N17Ras, N17Rac1, c-Jun, or treatment with a specific p38-K inhibitor. The critical function of this signaling cascade is indicated by prevention of Fas- or C sub(6)-ceramide-induced apoptosis after inhibition of Ras, Rac1, or JNK/p38-K.
    Keywords: Apoptosis ; Apoptosis ; Fas Antigen ; Ceramide ; Rac1 Protein ; Jnk Protein ; P38 Protein ; Gadd153 Protein ; Fas Antigen ; Gadd153 Protein ; Jnk Protein ; Rac1 Protein ; Ceramide ; P38 Protein;
    ISSN: 0021-9258
    E-ISSN: 1083-351X
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  • 4
    Language: English
    In: Journal of Biological Chemistry, 10/18/1996, Vol.271(42), pp.26389-26394
    Description: Fas induces apoptosis in lymphocytes via a poorly defined intracellular signaling mechanism. We and others have previously demonstrated the involvement and significance of a signaling cascade from the Fas receptor via sphingomyelinases and ceramide to Ras in apoptosis (Gulbins, E., Bissonette, R., Mahboubi, A., Nishioka, W., Brunner, T., Baier G., Baier-Bitterlich, G., Byrd, C., Lang, F., Kolesnick, R., Altman, A., and Green, D. (1995) Immunity 2, 341; Cifone, M. G., DeMaria, R., Roncali, P., Rippo, M. R., Azuma, M., Lanier, L. L., Santoni, A., and Testi, R. (1994) J. Exp. Med. 180, 1547-1552; Gill, B. M., Nishikata, H., Chan, G., Delovitch, T. L., and Ochi, A. (1994) Immunol. Rev. 142, 113-126). Here, we demonstrate an activation of the small G-proteins Rac 1 and Rac 2 after Fas receptor triggering. Expression of a transdominant inhibitory Ras mutant (N17Ras) prevents Rac 1 and Rac 2 stimulation, suggesting a signaling cascade from the Fas receptor via Ras to Rac 1 and Rac 2. Genetic and pharmacological inhibition of Ras or Rac 1 and Rac 2 stimulation blocks Fas-induced apoptosis, pointing to an important function of a Ras and Rac protein-regulated signaling pathway in Fas-mediated programmed cell death.
    Keywords: Chemistry ; Anatomy & Physiology;
    ISSN: 0021-9258
    E-ISSN: 1083-351X
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  • 5
    Language: English
    In: Cellular Physiology and Biochemistry, February 2011, Vol.27(1), pp.45-54
    Description: The preclinical compounds Bay 11-7082 and parthenolide trigger apoptosis, an effect contributing to their antiinflammatory action. The substances interfere with the activation and nuclear translocation of nuclear factor NFĸB, by inhibiting NFĸB directly (parthenolide) or by interfering with the inactivation of the NFĸB inhibitory protein IĸB-α (Bay 11-7082). Beyond that, the substances may be effective in part by nongenomic effects. Similar to apoptosis of nucleated cells, erythrocytes may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure, and cell shrinkage. Thus, erythrocytes allow the study of nongenomic mechanisms contributing to suicidal cell death, e.g. Ca2+ leakage or glutathione depletion. The present study utilized Western blotting to search for NFĸB and IĸB-α expression in erythrocytes, FACS analysis to determine cytosolic Ca2+ (Fluo3 fluorescence), phosphatidylserine exposure (annexin V binding), and cell volume (forward scatter), as well as an enzymatic method to determine glutathione levels. As a result, both NFĸB and IĸB-α are expressed in erythrocytes. Targeting the NFĸB pathway by Bay 11-7082 (IC50 ≈ 10 µM) and parthenolide (IC50 ≈ 30 µM) triggered suicidal erythrocyte death as shown by annexin V binding and decrease of forward scatter. Bay 11-7082 treatment further increased intracellular Ca2+ and led to depletion of reduced glutathione. The effects of Bay 11-7082 and parthenolide on annexin V binding could be fully reversed by the antioxidant N-acetylcysteine. In conclusion, the pharmacological inhibitors of NFĸB, Bay 11-7082 and parthenolide, interfere with the survival of erythrocytes involving mechanisms other than disruption of NFĸB-dependent gene expression.
    Keywords: Original Paper ; Cell Volume ; Eryptosis ; Calcium ; Phosphatidylserine Exposure ; Glutathione ; N-Acetylcysteine ; Biology ; Chemistry
    ISSN: 1015-8987
    E-ISSN: 1421-9778
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  • 6
    Language: English
    In: FEBS Letters, 1997, Vol.417(3), pp.301-306
    Description: The interaction of the CD40 receptor with its ligand has been shown to be crucial for the activation of B-lymphocytes. Here, we provide evidence that the pg39 molecule/CD40 ligand (gp39/CD40L) also functions as a stimulatory molecule for T-lymphocytes. Activation of T-lymphocytes via gp39/CD40L induced a strong activation of Jun-N-terminal kinase (JNK) and p38-K. Activation of these kinases correlates with a stimulation of Rac1 and inhibition of Rac1 prevents gp39/CD40L triggered JNK/p38-K activation. Further, cellular stimulation via the CD40 ligand results in tyrosine phosphorylation of cellular proteins and the activation of p56(lck). Inhibition of src-like kinases inhibits Rac1 as well as JNK/p38-K stimulation suggesting a signalling cascade from the gp39/CD40L via p56(lck) and Rac1 to JNK/p38-K.
    Keywords: Cd40 Ligand ; Gp39 ; T-Lymphocytes ; Tyrosine Kinase ; Jun-N-Terminal Kinase ; Biology ; Chemistry ; Anatomy & Physiology
    ISSN: 0014-5793
    E-ISSN: 1873-3468
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  • 7
    Language: English
    In: BBA - General Subjects, 2002, Vol.1572(1), pp.67-76
    Description: The transfection of mammalian cells by endocytosis is frequently hampered by low efficiency. To identify the bottlenecks, a system that allows the analysis of the intracellular pathway of DNA along the endocytic compartments in the eukaryote Saccharomyces cerevisiae was developed. DNA uptake in yeast cells was achieved by endocytosis when the cells were incubated with episomal DNA in the presence of 34% sucrose and subsequently shifted to a hypotonic medium to induce osmotical lysis of accumulated endocytic intermediates. The compartments of the endocytic pathway after the intersection of the endocytic and the vacuolar sorting pathway are preferred sites of DNA degradation. Either a transport blockade before this point or, even better, the presence of chloroquine, which is known as an adjuvant for transfection in mammalian cells, are required for successful transfection. The transport blockade can be achieved by deleting a GTPase of the endocytic pathway, Ypt51p, or ethanol. Chloroquine affects the compartments of the late endocytic pathway, and no effect is seen on transfection in a strain that is defective for YPT51 and accumulates the DNA in the early endocytic intermediates. To our knowledge, this is the first report on endocytic DNA uptake in S. cerevisiae .
    Keywords: Saccharomyces Cerevisiae ; Transfection ; Endocytosis ; Osmotic Lysis ; Chloroquine ; Biology ; Chemistry
    ISSN: 0304-4165
    E-ISSN: 1872-8006
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  • 8
    Language: English
    In: Journal of Molecular Biology, 1996, Vol.259(4), pp.704-717
    Description: The crystal structure of a lipase from the bacterium Chromobacterium viscosum ATCC 6918 (CVL) has been determined by isomorphous replacement and refined at 1.6 angstrom resolution to an R-factor of 17.8%. The lipase has the overall topology of an alpha / beta type protein, which was also found for previously determined lipase structures. The catalytic triad of the active center consists of the residues Ser87, Asp263 and His285. These residues are not exposed to the solvent, but a narrow channel connects them with the molecular surface. This conformation is very similar to the previously reported closed conformation of Pseudomonas glumae lipase (PGL), but superposition of the two lipase structures reveals several conformational differences, r.m.s. deviations greater than 2 angstrom are found for the C super( alpha )-atoms of the polypeptide chains from His15 to Asp28, from Leu49 to Ser54 and from Lys128 to Gln158. Compared to the PGL structure in the CVL structure, three alpha -helical fragments are shorter, one beta -strand is longer and an additional antiparallel beta -sheet is found. In contrast to PGL, CVL displays an oxyanion hole, which is stabilized by the amide nitrogen atoms of Leu17 and Gln88, and a cis-peptide bond between Gin291 and Leu292. CVL contains a Ca super(2+), like the PGL, which is coordinated by four oxygen atoms from the protein and two water molecules.
    Keywords: Chromobacterium Viscosum; Triacylglycerol-Hydrolase (Lipase); X-Ray Crystallography Pseudomonadaceae; Oxyanion ; Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 9
    Language: English
    In: Blood, 23 June 2016, Vol.127(25), pp.3281-90
    Description: Reduced-intensity conditioning has improved survival after hematopoietic stem cell transplantation (HSCT) for hemophagocytic lymphohistiocytosis (HLH) at the cost of more frequent mixed chimerism. The minimum level of donor chimerism (DC) required to prevent HLH reactivation in humans remains to be determined. In a multicenter retrospective study, 103 patients transplanted for hereditary HLH (2000-2013) and DC permanently or transiently 6 months (median, 5.1; range, 1.1-10 years) without reactivation. A second HSCT was performed in 18 patients (median, DC 4%; range, 0-19%). Death from reactivation occurred in 4 patients (22% of recurrences). Six patients died of transplant complications following a second HSCT (33% of second HSCT). We conclude that a DC 〉20%-30% is protective against late reactivation. Lower levels do not, however, inescapably result in recurrences. The decision for or against second HSCT must be based on a thorough risk assessment.
    Keywords: Chimerism ; Tissue Donors ; Lymphohistiocytosis, Hemophagocytic -- Immunology
    E-ISSN: 1528-0020
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  • 10
    Language: English
    In: Cellular Physiology and Biochemistry, 2003, Vol.13(6), pp.337-346
    Description: In nucleated cells cellular taurine is released prior to DNA fragmentation and the breakdown of phosphatidylserine asymmetry within the plasma membrane. Similar to what is seen in nucleated cells, phosphatidylserine asymmetry is also abolished in erythrocytes exposed to osmotic shock or oxidative stress. The present study has been performed to explore the sensitivity of erythrocytes from a taurine transporter knockout mouse (taut-/-) against osmotic shock and oxidative stress. Erythrocyte cell volume was estimated from forward scatter and breakdown of phosphatidylserine asymmetry was identified by determination of annexin binding using FACS analysis. Erythrocytes from taut-/- mice were compared to erythrocytes from wild type littermates (taut+/ +). Plasma concentration and erythrocyte content of taurine was significantly lower in taut-/- than in taut+/ + mice, but the intraerythrocyte taurine concentration did not exceed the plasma concentration. Hyperosmotic shock (exposure to 700 mOsm) and oxidative stress (exposure to 0.1 mM tert-butyl-hydroperoxide) significantly decreased the cell volume and increased the number of annexin binding sites of erythrocytes from both, taut-/- and taut+/ + mice. However, decrease of cell volume and increase of annexin binding was significantly blunted in erythrocytes from taut-/- mice as compared to their taut+/ + littermates. Stimulation of erythropoiesis by prior hemorrhage did not abrogate the difference between taut+/ + and taut-/- erythrocytes. The present observations point to a decreased sensitivity of mature erythrocytes from taut-/- mice to osmotic shock and oxidative stress, rendering them more resistant to apoptosis.
    Keywords: Original Paper ; Biology ; Chemistry
    ISSN: 1015-8987
    E-ISSN: 1421-9778
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