Zytophotometrische Bestimmung des Histon- und Gesamtproteingehalts von Zellen des lymphatischen Gewebes

Résumé L'étude au microscope électronique a révélé la présence de particules B dans le thymus de la souris NZB, adulte et saine. Les particules se forment dans un type de cellule thymique bien défini, la cellule réticulo-épithéliale à vacuoles et naissent par bourgeonnement des membranes plasmatiques limitant les vacuoles. L'élaboration de particules B dans lethymus d'animaux apparemment sains est un fait nouveau dont la signification n'est pas encore connue.

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Summary An electron microscopical study revealed the presence of virus-like particles in the thymus of adult healthy NZB-mice. The particles are found in the vacuoles of reticuloepithelial cells and are elaborated by a budding process from the membranes surrounding the vacuoles. The formation of B-particles in the thymus of apparently healthy animals is a so far unknown fact the significance of which remains to be determined.

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Avec l'aide du Centre National de la Recherche Scientifique et de l'Institut National de la Santé et de la Recherche Médicale, et la collaboration technique dévouée de M^lle J. Patry et de Mr. B. Fontaine.     

Hyaluronan and Its Receptors as Regulatory Molecules of the Endothelial Interface

On the surface of endothelial cells (ECs) lies the glycocalyx, a barrier of polysaccharides that isolates the ECs from the blood. The role of the glycocalyx is dynamic and complex, thanks to not only its structure, but its vast number of components, one being hyaluronan (HA). HA is a critical component of the glycocalyx, having been found to have a wide variety of functions depending on its molecular weight, its modification, and receptor–ligand interactions. As HA and viscous blood are in constant contact, HA can transmit mechanosensory information directly to the cytoskeleton of the ECs. The degradation and synthesis of HA directly alters the permeability of the EC barrier; HA modulation not only alters the physical barrier but also can signal the initiation of other pathways. EC proliferation and angiogenesis are in part regulated by HA fragmentation, HA-dependent receptor binding, and downstream signals. The interaction between the CD44 receptor and HA is a driving force behind leukocyte recruitment, but each class of leukocyte still interacts with HA in unique ways during inflammation. HA regulates a diverse repertoire of EC functions.     

Vergleichende morphologische und zytophotometrisch-autoradiographische Untersuchung der menschlichen Erythropoiese

Zusammenfassung Um die Abhängigkeit der erythropoietischen Zellformen von ihrer Stellung im Generationszyklus (G₁, S und G₂) zu untersuchen, wurde das Knochenmark von vier Normalpersonen mit der Kombination von zytophotometrischer DNS-Bestimmung (Feulgen-Photometrie) und autoradiographischer Technik mit ³H-TdR (in vitro) untersucht. Die Zellen wurden vor dieser Untersuchung in den nach Pappenheim gefärbten Ausstrichen differenziert.Sowohl in der Gruppe der basophilen Erythroblasten (E₁-E₃) als auch bei den polychromatischen Normoblasten (E₄) wurde eine postmitotische (G₁) und eine prämitotische Ruhephase (G₂) nachgewiesen. Beide Zellgruppen waren zu ca. 65% mit³H-TdR markiert (S).Unter den basophilen Erythroblasten war bei E₁ eine G₂-Phase, jedoch keine G₁-Phase nachweisbar. Demgegenüber fand sich bei E₃ eine ausgeprägte G₁-Phase, hingegen keine G₂-Phase. Bei E₂ war sowohl eine G₁-Phase als auch eine G₂-Phase erkennbar. Nach diesen Befunden stellen die Erythroblasten E₁-E₃ keine Zellgruppen mit jeweils vollständigem Generationszyklus dar, sondern sind als ein Zellkompartment zu verstehen, in dem die G₁-Phase vorwiegend durch die kleineren Zellen, die zytologisch die Merkmale der basophilen Normoblasten besitzen, und die G₂-Phase vorwiegend durch die größeren Zellen vom Typ der Proerythroblasten und Makroblasten repräsentiert wird.

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Comparative morphological and cytophotometric-autoradiographical investigation of erythropoiesis in the human

Summary In the different types of normal human nucleated red cells the stages of the cell cycle (G₁, S and G₂) were investigated by combined application of the cytophotometric determination of the DNA content (Feulgen photometry) and of autoradiographic labelling using ³H-TdR in vitro. The individual cells were identified in Pappenheim stain.In the basophilic erythroblasts (E₁-E₃) as well as in the polychromatic erythroblasts (E₄) about 62–65% of the cells were labelled (S). The unlabelled cells partly were diploid and partly were tetraploid, representing G₁ and G₂. The oxyphilic erythroblasts (E₅) mostly were diploid and unlabelled (G₁).Within the basophilic erythroblasts G₁ was demonstrated mainly in E₃, and G₂ was demonstrated mainly in E₁. In E₂, G₁ as well as G₂ were present. The results indicate that all basophilic erythroblasts belong to one cell compartment, in which G₁ is represented by the smaller cells commonly subclassified as basophilic normoblasts and G₂ is represented by the large cells usually called proerythroblasts and macroblasts.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

The relationship between the electromyogram-amplitude and isometric extension torques of neck muscles at different positions of the cervical spine

A group of 12 healthy men volunteered for the experiment. Electromyograms (EMG) were obtained from semispinalis capitis, splenius capitis, levator scapulae, and trapezius muscles. The flexion angle of the cervical spine was precisely adjusted to 0°, 10°, 20°, and 30° relative to the horizontal, with a constant angle of the atlanto-occipital joint. The subjects made eight short (about 2 s) vertical extension forces (6%, 12%,18%, 24%, 30%, 36%, 42%, and 48% of maximal voluntary peak contraction force). For each position, the centre of pressure under the head was determine as the basis for the calculation of the external lever arm. The presence of motor endplate regions was ascertained by multiple surface electrodes. The slopes of individual linear regression lines for the root mean square (rms)-values were dependent on the existence of endplates in the area of the electrodes — endplates caused smaller rms values per Newton metres of external torque. Significant intersubject differences between regression equations could not be eliminated by the normalization of EMG-parameters and/or torques. The elimination of gravity, the continuous monitoring of positions, and the consideration of localization of motor endplate regions were essential prerequisites for the acquisition of reliable relationships between EMG of different neck muscles and external torques. Two important conclusions were derived for the prediction of torques from EMG measurements: firstly, individual regression equations which take into account the position of the head and neck should be used; secondly, normalization procedures do not justify the application of average regressions to a group of subjects.     

Aldosterone increases kidney tubule cell oxidants through calcium-mediated activation of NADPH oxidase and nitric oxide synthase

Chronic hyperaldosteronism has been associated with an increased cancer risk. We recently showed that aldosterone causes an increase in cell oxidants, DNA damage, and NF-κB activation. This study investigated the mechanisms underlying aldosterone-induced increase in cell oxidants in kidney tubule cells. Aldosterone caused an increase in both reactive oxygen and reactive nitrogen (RNS) species. The involvement of the activation of NADPH oxidase in the increase in cellular oxidants was demonstrated by the inhibitory action of the NADPH oxidase inhibitors DPI, apocynin, and VAS2870 and by the migration of the p47 subunit to the membrane. NADPH oxidase activation occurred as a consequence of an increase in cellular calcium levels and was mediated by protein kinase C. The prevention of RNS increase by BAPTA-AM, W-7, and L-NAME indicates a calcium-calmodulin activation of NOS. A similar pattern of effects of the NADPH oxidase and NOS inhibitors was observed for aldosterone-induced DNA damage and NF-κB activation, both central to the pathogenesis of chronic aldosteronism. In summary, this paper demonstrates that aldosterone, via the mineralocorticoid receptor, causes an increase in kidney cell oxidants, DNA damage, and NF-κB activation through a calcium-mediated activation of NADPH oxidase and NOS. Therapies targeting calcium, NOS, and NADPH oxidase could prevent the adverse effects of hyperaldosteronism on kidney function as well as its potential oncogenic action.     

Development and validation of a highly efficient protocol of porcine somatic cloning using preovulatory embryo transfer in peripubertal gilts

The efficiency of porcine somatic nuclear transfer (born piglets/transferred embryos) is low. Here, we report a highly efficient protocol using peripubertal gilts as recipients synchronized to ovulate approximately 24 h after transfer of cloned embryos. Retrospectively, we compared the efficiency of two different synchronization protocols: In group 1, recipient animals were synchronized to ovulate approximately 6 h prior to surgical embryo transfer while in group 2 the animals were treated to ovulate 24 h after embryo transfer. In total, 1562 cloned embryos were transferred to 12 recipients in group 1; two of them became pregnant (16.7%). One pregnancy was lost on day 32, the second pregnancy went to term, and led to the birth of one healthy piglet after Cesarean section. In group 2, 1531 cloned embryos were transferred to 12 recipients. Nine recipients (75.0%) became pregnant as determined by ultrasound scanning on day 25. All pregnancies went to term and delivered a total of 47 live-born piglets. The cloning efficiency of both groups differed significantly (group 1: 0.1%, group 2: 3.1%, p < 0.05). This modified protocol was then applied in subsequent experiments using different types of transgenic and nontransgenic donor cells with similar success rates. Results show that this protocol is robust and highly reproducible, and can thus be employed for routine production of cloned pigs.     

Plasminogen activator inhibitor type 1 inhibits smooth muscle cell proliferation in pulmonary arterial hypertension

RATIONALE: Pulmonary arterial smooth muscle cells (PASMCs) in the medial layer of the vessel wall are responsible for vessel homeostasis, but also for pathologic vascular remodelling in diseases, such as idiopathic pulmonary arterial hypertension (IPAH). Vascular remodelling in IPAH results in vessel stiffness, occlusion, and increased vascular resistance, but its underlying mechanisms remain to be fully elucidated. In this study, we investigated the expression and function of plasminogen activator inhibitor (PAI)-1, an inhibitor of the plasminogen activator system and target gene of the transforming growth factor (TGF)-beta1 signalling cascade, in PASMC in IPAH. METHODS AND RESULTS: RNA and protein analysis from lung tissues of donors and patients with IPAH (n=7 each) revealed a significant downregulation of PAI-1 in IPAH lungs. Immunohistochemical analysis localised PAI-1 to the bronchial and alveolar epithelium, as well as to vascular and airway smooth muscle cells. PAI-1 was also downregulated in primary PASMC derived from IPAH lungs as compared with donor-derived PASMC. In order to elucidate PAI-1 function, primary PASMC were stimulated with active recombinant (r)PAI-1, or transfected with PAI-1-specific siRNA. Stimulation with rPAI-1 led to decreased PASMC proliferation and adhesion to vitronectin, and increased PASMC migration. In contrast, PAI-1 knock-down with siRNA increased PASMC proliferation and decreased PASMC migration. CONCLUSIONS: PAI-1 is significantly downregulated in PASMC in IPAH, on the mRNA and protein level. PAI-1 negatively regulates PASMC proliferation, while it increases PASMC migration. Thus, its loss in IPAH may therefore contribute to pathologic vascular remodelling in IPAH.     

Identification of young megakaryocytes by immunofluorescence and cytophotometry.

The DNA-content of fluoresceine-labeled platelet antigen containing cells of mouse bone marrow was measured. For immunofluorescence highly specific anti-mouse-platelet-serum and fluoresceine-conjugated antigammaglobuline was used, applying the "sandwich" technique. Three hundred panoptically identifable megakaryocytes served as control group. The DNA-polyploidization pattern of megakaryocytes and immunofluorescence positive cells was almost identical. However, among the immunofluorescence positive cells a considerable amount of cells showed DNA-values lower than 4c, whereas the megakaryocytes of the Pappenheim stained smears revealed no DNA-values lower than 4c. The percentages of diploid and tetraploid cells, respectively, was 6 and 7% compared with 0 and 1% of panoptically identifiable megakaryoctyes. The results suggest that young megakaryocytic cells with diploid and tetraploid DNA-values can be detected by immunofluorescence technique, indicating that the flow from the uncommited to the committed megakaryocytic precursor cell appears at this early stage of megakaryocyte production.