Phosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress

Fluid shear stress activates the endothelial nitric oxide (NO) synthase (eNOS) by a mechanism which does not require an increase in the intracellular concentration of free Ca2+ ([Ca2+]i), and is sensitive to several kinase inhibitors. Although phosphorylation of eNOS has been suggested to regulate enzyme activity, the mechanism of eNOS activation is still unclear. Here we demonstrate that fluid shear stress elicits the phosphorylation of eNOS on tyrosine and serine residues. Inhibition of phosphatidylinositol 3-kinase (PI3K), using wortmannin or a dominant negative mutant of its downstream target, Akt (protein kinase B), prevented the maintained serine phosphorylation and activation of eNOS. Enhancing eNOS phosphorylation by inhibiting serine/threonine phosphatases, increased eNOS activity by approximately twofold, as assessed by the accumulation of intracellular cyclic GMP, without increasing the intracellular concentration of free Ca2+. These data suggest that shear stress activates a pathway involving PI3K and the serine/threonine kinase Akt, which phosphorylates eNOS. This phosphorylation directly increases eNOS activity at resting [Ca2+]i, thus rendering the shear stress-induced activation of eNOS apparently Ca2+-independent.     

Somatotopic organization along the central sulcus, for pain localization in humans, as revealed by positron emission tomography

Regional cerebral blood flow was measured with positron emission tomography (PET) in six healthy volunteers at rest and during experimentally induced, sustained cutaneous pain on the dorsum of the right hand or on the dorsum of the right foot. Pain was inflicted by intracutaneous injection of capsaicin, providing a mainly C-fibre nociceptive stimulus. Statistical analysis showed significant activations along the central sulcus (SI) area when comparing pain in the hand to pain in the foot. Separate comparison of both pain states to a baseline revealed different locations along the central sulcus for hand pain and foot pain. The encountered differences are consistent with what is previously known about the somatotopics of non-painful stimuli. When comparing painful stimuli to baseline, the contralateral anterior cingulate gyrus, the ipsilateral anterior insular cortex and the ipsilateral prefrontal cortex were implicated. The results are consistent with an involvement of SI in the spatial discrimination of acute cutaneous pain. Received: 17 October 1996 / Accepted: 12 May 1997     

Enhancement of death-receptor induced caspase-8-activation in the death-inducing signalling complex by uncoupling of oxidative phosphorylation

Signalling through the death receptor CD95 induces apoptosis by formation of a signalling complex at the cell membrane and subsequent caspase-8 and caspase-3-activation. Treatment of Jurkat T cells with protonophores across the mitochondrial membrane such as 2,4-dinitrophenol (DNP) enhances the death-inducing capacity of CD95. In this study, we show that this enhancement is due to the specific acceleration of caspase-8-processing and activation at the CD95-receptor. DNP-treatment did not affect NF-kappaB-induction by CD95. Immunoprecipitation experiments showed that the amounts of the adapter FADD/MORT1 and pro-caspase-8 at the CD95-receptor were not altered by DNP. Subcellular fractionation studies revealed that the amount of mature caspase-8 but not pro-caspase at the membrane was increased following CD95-stimulation in the presence of DNP. As a consequence of caspase-activation, c-FLIP-levels in the cytosol decreased. In Jurkat cells overexpressing c-FLIPS, DNP was still able to enhance caspase-activation. The enhancing capacity of DNP was seen in some cell lines (Jurkat, CEM and HeLa) but not in SKW6 cells and was also found in mitogen-stimulated human T cells. Furthermore, the enhancement extended to TRAIL-induced caspase-activation. Thus, a mechanism exists by which caspase-8-activation can be accelerated at death receptors and this mechanism can be triggered by targeting mitochondrial oxidative phosphorylation.     

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFα, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFα and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.     

Detection of a mucin marker for the adenoma-carcinoma sequence inhuman colonic mucosa by monoclonal antibody AM-3.

The monoclonal antibody AM-3 was raised against mucins extracted from human colorectal carcinomas. It reacted strongly with sections of paraffin wax embedded colorectal carcinoma. In colonic adenoma tissue the percentage of cells expressing the epitope detected by AM-3 correlated with the degree of dysplasia. In contrast to immunohistochemical staining, which did not show the presence of the antigen in histologically normal mucosa, the more sensitive enzyme linked immunosorbent assay (ELISA) and immunoblot assays showed that it was weakly expressed in this tissue. AM-3 reacted with variable frequency with several normal and malignant human tissues, indicating that the detected epitope is not restricted to colonic tissue. In colonic carcinomas it is present on a sialomucin of apparent relative molecular mass of more than 440,000. These data suggest that the antigen detectable with AM-3 may be useful in the assessment of premalignant changes in colonic adenomas.     

Histone H1-mediated transfection: role of calcium in the cellular uptake and intracellular fate of H1-DNA complexes

Previously, we have shown that the transgene expression in the endothelial cell line ECV 304 strongly depends on the presence of low concentrations of Ca2+. However, it remained unclear, which transfection steps are controlled by Ca2+ ions. In the present study, we constructed transfection complexes of digoxigenin-labelled DNA and FITC-labelled histone H1. We monitored the pathway of these complexes with the use of anti-digoxigenin and anti-cathepsin B antibodies and immunofluorescence microscopy. Double labelling of DNA and cathepsin B permitted the localization of transfection complexes into endosomes/lysosomes which suggests an uptake of transfection complexes via endocytosis. It was also found that the uptake of transfection complexes by the cells was independent of the presence or absence of Ca2+ ions in the transfection medium. On the other hand, the presence of Ca2+ in the transfection medium dramatically changed the composition of the transfection complexes inside the endosome/lysosome compartment, which resulted in a strong reduction of H1 binding to DNA. Presence of Ca2+ in the postincubation medium for 24 h resulted in release of the transfection complexes with reduced H1 content from the endosomes/lysosomes into the cytosol. In the absence of Ca2+ the transfection complexes practically disappeared. These results allow us to come to the following conclusions: Ca2+ ions control the reorganization of the transfection complexes in endosomes/lysosomes and their release into the cytosol, which is an important prerequisite for transgene expression, whereas uptake of transfection complexes by the cells is not dependent on Ca2+.     

Feinstrukturell-morphometrische Befunde an der Kammerwand hypertrophierter menschlicher linker Ventrikel

Zusammenfassung An den KammerwÄnden menschlicher linker Ventrikel, die auf Grund einer Aortenstenose, einer Aorteninsuffizienz oder eines kombinierten Aortenvitium hypertrophiert waren, wurden licht- und elektronenmikroskopisch morphometrische Untersuchungen angestellt. Die Ergebnisse wurden mit denen, die an nicht belasteten menschlichen linken Ventrikeln gewonnen wurden, verglichen.Lichtmikroskopisch unterscheiden sich die Anteile der Volumendichten des Interstitium und der Herzmuskelzellen am gesamten Herzmuskelgewebe nicht statistisch signifikant. Es konnte morphometrisch eine Zellvergrö\erung festgestellt werden, die aus der signifikanten Verringerung der Volumendichte der Zellkerne (P<0,001) und der Anzahl der Zellkerne pro TestflÄche (P<0,0001) gegenüber den beiden Normalkollektiven resultiert. Elektronenmikroskopisch ist eine Zunahme der Volumendichten der Myofibrillen (P<0,0001) auf Kosten des restlichen Cytoplasmas (P<0,001) festzustellen, wÄhrend die Volumendichte der Mitochondrien im Vergleich mit den jungen und alten Patienten abnahm (P<0,0001). Die OberflÄchendichte der Mitochondrien verringerte sich gegenüber den beiden Vergleichskollektiven (P<0,001) ebenso wie die der Cristae mitochondriales (P<0,0001). Diese Ergebnisse finden ihr morphologisches Korrelat in Mitochondriendestruktionen. Eine vermehrte Myolyse hat bei den hypertrophierten Herzen, die alle gewichtsmÄ\ig über dem kritischen Herzgewicht lagen, noch nicht eingesetzt. Bei allen Patienten wurde der herzchirurgische Eingriff mit Erfolg durchgeführt.

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Ultrastructural morphometric analysis of hypertrophied human myocardial left ventricles

Summary Biopsies of hypertrophied human myocardial left ventricles were investigated morphometrically. The diagnoses of the patients were stenosis of the aortic valve, aortic insufficiency or a combination of both lesions. The results were compared with those from normally loaded human left ventricles.There are no differences on light microscopical level between the volume densities of interstitial tissue and of heart muscle cells in the three groups of patients. A significant diminution of the volume density of the nuclei (P<0.001) and the number of nuclei per test area (P<0.0001) when compared with normal groups suggests an increase in volume of the single heart muscle cell. The ultrastructural study shows marked increase in volume density of myofibrils (P<0.0001), with accompanying decrease in the volume densities of mitochondria (P<0.0001) and the remaining cytoplasm (P<0.001). A gross decrease in the surface area of mitochondria (P<0.001) and of cristae mitochondriales (P<0.0001) is found. The morphological equivalents of this result are numerous stages of mitochondrial destruction including cristolysis. All myocardial weights were beyond the critical heart weight.

Mit dankenswerter Unterstützung der Deutschen Forschungsgemeinschaft über den Sonderforschungsbereich SFB 104     

IR-und UV-spektroskopische Eigenschaften einer homologen Reihe von molekulareinheitlichen, 12gliedrige Ringe enthaltenden Leiteroligomeren

The reactions of multiple acrylates of oligo[(hydroxyphenylene)methylene] s ( 2a–2f ), strongly diluted in boiling benzene, with 2,2′-azoisobutyronitrile in a mole ratio of 1:10, were investigated. The elemental analysis and the determination of relative molar masses of the resulting products ( 3, 4, 5, 6a, 6b, 7a and 7b ) demonstrate that their molecules contain two nitrile groups. These data, together with the IR and UV spectra, show that the products are molecularly uniform ladder oligomers with two 1-cyano-1-methylethyl groups on both ends.