Die Ultrastruktur der Kapillarwand in der menschlichen Placenta zur Zeit der Schwangerschaftsmitte

Zusammenfassung Die Wand der Kapillaren in der menschlichen Placenta aus der Schwangerschaftsmitte wird elektronenmikroskopisch untersucht und zu der Wand des Sinusoides der reifen Placenta in Beziehung gesetzt. Bereits zur Zeit der Schwangerschaftsmitte sind vereinzelt Sinusoide nachweisbar, doch treten sie gegenüber den Kapillaren zahlenmäßig in den Hintergrund.Die Kapillaren des Zottenbinnenraumes besitzen keine Basalmembran; sie sitzen meist, nur durch einen Spalt getrennt, einer Pericytenschicht auf. Die Pericyten haben häufig fußförmige Ausläufer, die die Basalmembran des Cytotrophoblasten erreichen. Die Kapillarendothelien sind zwar einreihig angeordnet, überlappen aber einander in ausgedehnter Weise.Im Cytoplasma der Kapillarendothelien findet man häufig eine feinfilamentäre Zeichnung, jedoch nur nach Kaliumpermanganat-Kontrastierung.Die Kapillaren der unreifen Placenta sind durch das Fehlen der Basalmembran, durch die ungewöhnliche Dicke und durch die starke Überlappung ihres Endothels für eine Gefügedilatation besonders geeignet.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Optimization of culture conditions for human corneal endothelial cells

Summary Long-term cultivation of human corneal endothelial cells (HCEC) was optimized with respect to different components of the culture system: 25 different nutrient media, different sera, 6 mitogens and various substrates were tested in their ability to influence clonal growth and morphology of HCEC. F99, a 1∶1 mixture of the two media M199 and Ham’s F12, was the most effective basal medium in promoting clonal growth of HCEC. Among various sera, human serum and fetal bovine serum showed optimal growth promoting activities in combination with F99, whereas newborn bovine serum (NBS) was by far superior for the development of a typically corneal endothelial morphology. Crude fibroblast growth factor (FGF), or alternatively endothelial cell growth supplement, was absolutely essential for clonal growth of HCEC at low serum concentrations, for example 5% NBS. Formation of a monolayer with a morphology similar to corneal endothelium in vivo was observed only on culture dishes coated with basal membrane components such as collagen type IV, laminin, or fibronectin. The most pronounced effect on morphologic appearance was obtained by culturing the cells on the extracellular matrix (ECM) produced by bovine corneal endothelial cells. Moreover, ECM could substitute for crude FGF in clonal growth assays.     

Differential regulation of expression of two platelet-derived growth factor receptor subunits by transforming growth factor-beta

The binding of three radiolabeled isoforms of platelet-derived growth factor (PDGF), 125I-PDGF-AA, 125I-PDGF-AB, and 125I-PDGF-BB, is differentially affected by exposure of quiescent 3T3 cells to transforming growth factor-beta (TGF-beta). By 24 h after exposure to TGF-beta, binding of 125I-PDGF-AA and 125I-PDGF-AB is almost completely lost, whereas binding of 125I-PDGF-BB is reduced by only 40%. The loss of PDGF-binding sites caused by TGF-beta is time- and concentration-dependent and reflects a change in the pattern of expression of receptor subunits; the number of alpha-subunits decreases, and the number of beta-subunits increases. The loss of binding sites for PDGF-AA is accompanied by a decreased mitogenic response to PDGF-AA but not to PDGF-AB or PDGF-BB. These results suggest that TGF-beta may differentially regulate the expression of PDGF-binding sites and the mitogenic responsiveness toward the three PDGF isoforms. TGF-beta did not stimulate synthesis of PDGF A-chain mRNA or PDGF-AA protein, and PDGF-AA receptors could not be restored by the presence of suramin, suggesting that the loss of binding sites may result from direct effects on receptor expression rather than autocrine down-regulation by PDGF-AA.     

Analysis by high-resolution two-dimensional electrophoresis of differentiation-dependent alterations in cytosolic protein pattern of HL-60 leukemic cells.

HL-60 leukemic cells were differentiated along the neutrophilic pathway with retinoic acid (RA) or along the monocytic pathway with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Using a high-resolution two-dimensional electrophoresis technique and subsequent silver staining, differentiation-dependent changes in cytosolic protein pattern of HL-60 cells were analysed and were compared with the cytosolic protein pattern of human neutrophils. The amount of 64 and 50 out of a total of 632 proteins studied was increased or decreased in RA- and 1,25(OH)2D3-differentiated HL-60 cells, respectively, in comparison to undifferentiated HL-60 cells. Thirty-three of these proteins were similarly altered in RA- and 1,25(OH)2D3-differentiated HL-60 cells. Twenty-two and 25 of the proteins altered in amount in RA- or 1,25(OH)2D3-differentiated HL-60 cells versus undifferentiated HL-60 cells were similarly altered in human neutrophils in comparison to undifferentiated HL-60 cells. Seven and 10 of the proteins altered in amount in RA- or 1,25(OH)2D3-differentiated HL-60 cells had specific equivalents in neutrophil cytosol. Our results show (i) that neutrophilic and monocytic differentiation is associated with decreases and increases in amount of cytosolic proteins; (ii) that both differentiation processes share a common set of alterations; and (iii) are associated with specific alterations in protein amount.     

Expression of the E. coli nadB gene and characterization of the gene product L-aspartate oxidase

Quinolinic acid is synthesized in E. coli by the enzymes L-aspartate oxidase and quinolinate synthase A, the genes of which are named nadB and nadA. In our previous work we cloned and characterized the two genes (Flachmann, R., Kunz, N., Seifert, J., Gütlich, M., Wientjes, F.J., L?ufer, A. & Gassen, H.G. (1988) Eur. J. Biochem. 175, 221-228). Here we report on the expression of the nadB gene under control of the inducible left promoter of the bacteriophage lambda. The yield of the active gene product L-aspartate oxidase was enhanced up to 20% of the soluble cell protein. The enzyme was purified to homogeneity in a three-step procedure and the reading frame of the L-aspartate oxidase gene was confirmed by Edman degradation of five cyanogen bromide peptides. L-Aspartate oxidase shows no classical Michaelis-Menten behaviour but is subject to a substrate inactivation. The apparent Km values were different for substrate concentrations below and above 1mM and were determined to 0.5 mM and 4.1mM, respectively. The active form of the enzyme is a monomer of 60,284 Da and contains one molecule of FAD and nine cysteine residues, four of which built up two disulfide bonds. The isoelectric point of the protein was determined to be at pH 5.6. Chemical modifications of the enzyme showed that at least one tyrosine and one histidine residue are essential for enzyme activity. The coenzyme-binding domain is located in the amino-terminal part of the polypeptide chain as revealed by a sequence comparison to other dinucleotide binding enzymes. Furthermore, there is evidence for a relationship to fumarate reductase and succinate dehydrogenase of E. coli.     

Regulatory elements controlling expression of the Drosophila homeotic gene fork head.

The region-specific homeotic gene fork head (fkh) is expressed and required in a variety of tissues of the developing Drosophila embryo. In order to identify the cis regulatory elements directing the complex spatio-temporal expression pattern of fkh, we have studied the subpatterns directed by defined fragments of fkh genomic DNA. These experiments enabled us to distinguish separate regulatory elements specific for the different expression domains of fkh. In addition, our analysis revealed several unexpected features such as the redundancy of regulatory elements and the overlap of regulatory elements with the transcribed regions of other genes. Moreover, the separation of normally contiguous elements effecting expression in the posterior terminal fkh domain appears to lead to novel expression domains which do not correspond to known developmental units in the embryo.     

Primary structure and functional expression of a Drosophila cyclic nucleotide-gated channel present in eyes and antennae.

Cyclic nucleotide-gated (CNG) ion channels serve as downstream targets of signalling pathways in vertebrate photoreceptors and olfactory sensory neurons. Whether CNG channels subserve similar functions in invertebrate photoreception and olfaction is unknown. We have cloned genomic DNA and cDNA encoding a cGMP-gated channel from Drosophila. The gene contains at least seven exons. Heterologous expression of cloned cDNA in both Xenopus oocytes and HEK 293 cells gives rise to functional ion channels. The Drosophila CNG channel is approximately 50-fold more sensitive to cGMP than to cAMP. The voltage dependence of blockage by divalent cations is different compared with the CNG channel of rod photoreceptors, and the Ca2+ permeability is much larger. The channel mRNA is expressed in antennae and the visual system of Drosophila. It is proposed that CNG channels are involved in transduction cascades of both invertebrate photoreceptors and olfactory sensillae.     

Vertically stratified frugivore community composition and interaction frequency in a liana fruiting across forest strata

Vertical stratification is a key feature of tropical forests and structures plant–frugivore interactions. However, it is unclear whether vertical differences in plant-frugivore interactions are due to differences among strata in plant community composition or inherent preferences of frugivores for specific strata. To test this, we observed fruit removal of a diverse frugivore community on the liana Marcgravia longifolia in a Peruvian rain forest. Unlike most other plants, Marcgravia longifolia produces fruits across forest strata. This enabled us to study effects of vertical stratification on fruit removal without confounding effects of plant species and stratum. We found a high number of visits of a few frugivore species in the understorey and a low number of visits of many different frugivores in the canopy and midstorey. Whereas partial and opportunistic frugivores foraged across strata with differing frequencies, obligate frugivores were only found eating fruits in the higher strata. Avian frugivores foraging in the canopy were mainly large species with pointed wings, whereas under- and midstorey avian foragers were smaller with rounded wings. Our findings suggest a continuous shift in the frugivore community composition along the vertical gradient, from a few generalized frugivores in the understorey to a diverse set of specialized frugivores in the canopy. This shift in the frugivore community leads to correlated, reciprocal changes from specialized to generalized plant-frugivore interactions. Thus, we conclude that vertical niche differentiation between species in tropical forests persists even when food resources are available across strata. This highlights its role for promoting biodiversity and ecosystem functioning.     

Morphological parameters affecting false lumen thrombosis following type B aortic dissection: a systematic study based on simulations of idealized models

Type B aortic dissection (TBAD) carries a high risk of complications, particularly with a partially thrombosed or patent false lumen (FL). Therefore, uncovering the risk factors leading to FL thrombosis is crucial to identify high-risk patients. Although studies have shown that morphological parameters of the dissected aorta are related to FL thrombosis, often conflicting results have been reported. We show that recent models of thrombus evolution in combination with sensitivity analysis methods can provide valuable insights into how combinations of morphological parameters affect the prospect of FL thrombosis. Based on clinical data, an idealized geometry of a TBAD is generated and parameterized. After implementing the thrombus model in computational fluid dynamics simulations, a global sensitivity analysis for selected morphological parameters is performed. We then introduce dimensionless morphological parameters to scale the results to individual patients. The sensitivity analysis demonstrates that the most sensitive parameters influencing FL thrombosis are the FL diameter and the size and location of intimal tears. A higher risk of partial thrombosis is observed when the FL diameter is larger than the true lumen diameter. Reducing the ratio of the distal to proximal tear size increases the risk of FL patency. In summary, these parameters play a dominant role in classifying morphologies into patent, partially thrombosed, and fully thrombosed FL. In this study, we point out the predictive role of morphological parameters for FL thrombosis in TBAD and show that the results are in good agreement with available clinical studies.

     

Die Feinstruktur der Schweineaorta beim experimentellen Lathyrismus

Zusammenfassung Die Veränderungen der Aortenfeinstruktur, welche eine Vergiftung mit-Amino-Propio-Nitril (Lathyrusfaktor) bei jungen Schweinen hervorruft, werden beschrieben.Der Benninghoffsche Spannapparat der Media wird frühzeitig durch eine Lösung der muskulo-elastischen Kontinuität gelockert. Im Vordergrunde der morphologischen Veränderungen stehen dann Zerfallserscheinungen der elastischen Substanz, welche zu einer Desintegration der elastischen Lamellen sowie der elastischen interlamellären Schrägverbinder führen und endlich eine völlige Strukturauflösung der ganzen Mediaorganisation mit herdförmigen Totalnekrosen zur Folge haben. Das mit dem Mediafasersystem strukturell und funktioneil eng verbundene kollagen-elastische Fasergitter der Intima zerfällt; die subendotheliale Intima wird dabei hochgradig aufgetrieben durch eine Einlagerung von Ödemflüssigkeit und wahrscheinlich auch Blutserum. Die feinnetzig-zweiphasige Normaldarstellung der Grundsubstanz geht verloren. Die Feinstruktur der Kollagenfibrillen bleibt unverändert; es wird darauf hingewiesen, daß trotzdem eine schwere Schädigung auch des Kollagens anzunehmen ist. Die Mediamuskelzellen wandeln sich weitgehend in Fibroblasten um; die Langhans-Zellen der Intima werden in gleicher Weise aktiviert. Zellnekrosen treten in der Media nur dann auf, wenn sekundär die subakute Aortenwandverdickung eine Mangelsituation für den Zelleigenstoffwechsel hervorruft.In der Umgebung der Vasa vasorum kommt es zu kleinen Blutungen, die mit Fortschreiten der Mediazerstörung in große, dissezierende Blutungen übergehen können. Der wahrscheinlich durch die Elastizitätsverminderung der Media eingeleitete Mechanismus der Gefäßrupturierung wird beschrieben und mit dem vonGöre und vonDoerr vermuteten Mechanismus der Bildung von Aneurysmata dissecantia der menschlichen Aorta in Parallele gesetzt.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft. — FräuleinElke CarStensen schulden wir Dank für unermüdliche, gewissenhafte und geschickte technische Mitarbeit.