Über den submikroskopischen Bau des Grundhäutchens der Hirnkapillaren

Zusammenfassung Auf Grund der polarisationsoptischen Analyse besitzt das homogene Kapillar-Grundhäutchen eine geordnete Feinstruktur. Es ist ein Eiweiß-Lipoidsystem, in dem die Proteinkomponente als Träger- oder Gerüstsubstanz mengenmäßig überwiegt. Die Proteine zeigen teilweise eine fibrilläre Struktur mit geringer mizellarer Orientierung. In der Fläche der Membran haben die Fibrillen keine bevorzugte Verlaufsrichtung, sie liegen statistisch ungeordnet. Zu der Membranoberfläche verlaufen sie jedoch annähernd parallel, so daß eine folienartige Textur resultiert. In dieses Proteingerüst sind Lipoidmolekeln in radiärer Richtung orientiert eingelagert. Nach einer Näherungsrechnung dürfte es sich um eine bimolekulare Lamelle handeln, wahrscheinlich ist, daß die Lipoide inselförmig in das Proteingerüst eingesprengt sind. Die gefundene Struktur wird in Beziehung zu den Befunden neuerer Arbeiten über die Blut-Hirnschranke und die Blut-Liquorschranke gesetzt. Hierbei wird die Bedeutung der Membranlipoide für die Regulierung des Durchtritts lipoidlöslicher Stoffe, diejenige der Proteinlamellen für die Regulierung der Durchlässigkeit für Wasser und wasserlösliche Stoffe erörtert. Die Endothelmembran, d. h. das Grundhäutchen der Kapillare wird als das morphologische Substrat der Schrankenfunktion angesehen.Die Untersuchung wurde mit dankenswerter Unterstützung der Deutschen Forschungsgemeinschaft ausgeführt.     

A hierarchical Bayesian model to predict the duration of immunity to Haemophilus influenzae type b

A hierarchical Bayesian regression model is fitted to longitudinal data on Haemophilus influenzae type b (Hib) serum antibodies. To estimate the decline rate of the antibody concentration, the model accommodates the possibility of unobserved subclinical infections with Hib bacteria that cause increasing concentrations during the study period. The computations rely on Markov chain Monte Carlo simulation of the joint posterior distribution of the model parameters. The model is used to predict the duration of immunity to subclinical Hib infection and to a serious invasive Hib disease.     

Effect of cationic lipids in the formation of asymmetries in supported bilayers

We have studied the formation of a supported bilayer containing both cationic and zwitterionic lipids by fusion of small unilamellar vesicles (SUV) onto the solid surface at low salt conditions using a combination of attenuated total reflection infrared (ATR-IR) and deuterium NMR spectroscopy with microcalorimetry. The data suggest that a significant cationic lipid asymmetry between the outer (distal) and the inner (proximal) monolayer of a supported bilayer results under conditions of prolonged incubation times of the solid support with the SUV coating solution. For a SUV composition of DPPC/DHDAB (4:1) we observed an enrichment of the cationic component in the proximal monolayer of up to 200% compared to the distal monolayer after 12 h incubation. It is suggested that the electrostatic potential arising from the solid surface is the driving force for the creation of this asymmetry by means of directed flip-flop between the monolayers and/or by temporary fusion between SUV from the bulk with the supported bilayer.     

Adducts with haemoglobin and with DNA in epichlorohydrin-exposed rats

Epichlorohydrin (1-chloro-2,3-epoxypropane; ECH) is an important industrial chemical and a carcinogen in experimental animals. The main aims of the present study were to characterize the adduct formation in female Wistar rats and to identify adducts that could potentially be used in human biomonitoring studies. The total binding of radioactivity to haemoglobin in rats administered 0, 0. 11, 0.22, 0.43, or 0.97 mmol [3H]ECH/kg body weight by i.p. injection, and sacrificed 24 h after treatment, was linearly related to a dose up to 0.43 mmol/kg body weight. The binding at the highest dose was higher than predicted by extrapolation from lower doses, indicating saturation of a metabolic process for elimination of ECH. Ion-exchange chromatography of a globin hydrolysate showed one major radioactivity peak corresponding to S-(3-chloro-2-hydroxypropyl)cysteine. The half-life of this adduct was estimated as about 4 days by analysis of globin from rats administered 0.43 mmol/kg body weight and sacrificed after 1, 2 and 9 days. Crosslinking of the adduct, presumably with glutathione, appeared to be the predominant secondary reaction. Hydrolysis of N-(3-chloro-2-hydroxypropyl)valine, the primary reaction product of ECH with N-terminal valine, would give N-(2,3-dihydroxypropyl)valine. A sensitive gas chromatography/mass spectrometry method for the dihydroxypropyl adduct was used to follow its formation and removal after administration of nonlabelled ECH (0.11 mmol/kg body weight). The level of this adduct reached a maximum of about 20 pmol/g globin after a few weeks, corresponding to about 0.1% of the initial binding of ECH to globin. N-7-(3-Chloro-2-hydroxypropyl)guanine was detected in rats administered 0.97 mmol [3H]ECH/kg body weight and sacrificed 6 h after treatment. The adduct levels in haemoglobin and DNA were compared with previously reported adduct levels in male Fischer 344 rats exposed to propylene oxide. Despite its higher chemical reactivity, the capacity of ECH to alkylate macromolecules in vivo was found to be somewhat lower than that of propylene oxide.     

Attenuation of NF-kappaB signaling response to UVB light during cellular senescence

The ability of cells to adapt to environmental stresses undergoes a progressive reduction during aging. NF-kappaB-mediated signaling is a major defensive system against various environmental challenges. The aim of this study was to find out whether replicative senescence affects the response of the NF-kappaB signaling pathway to UVB light in human WI-38 and IMR-90 fibroblasts. The exposure of early passage fibroblasts to UVB light inhibited the proliferation and induced a flat phenotype similar to that observed in replicatively senescent fibroblasts not exposed to UVB light. The UVB radiation dose used (153 mJ/cm2) did not induce apoptosis in either early or late passage WI-38 fibroblasts. UVB exposure induced a prominent activation of the NF-kappaB signaling pathway both in early and in late passage WI-38 and IMR-90 fibroblasts. Interestingly, the response to UVB light was significantly attenuated in late passage fibroblasts. This attenuation was most prominent in DNA binding activities of nuclear NF-kappaB complexes. Similar senescence-related attenuation was also observed in the DNA binding activities of nuclear AP-1 and Sp-1 factors after UVB treatment. Immunoblotting and -cytochemistry showed an increase in nuclear localization of p50 and p65 components of NF-kappaB complexes. Supershift experiments showed that the specific NF-kappaB complexes contain p50 and p65 protein components but not p52 and c-Rel proteins. Cytoplasmic IkappaBalpha showed a marked decrease at protein level but an increase in phosphorylation after UVB treatment. Transient transfection assays with TK5-CAT and TK10-CAT plasmids carrying NF-kappaB-responsive sites of the TNFalpha promoter were used to analyze the functional activity of the NF-kappaB complexes. Results showed that UVB exposure induced an increase in NF-kappaB-driven CAT expression both in early and in late passage fibroblasts though the response was significantly stronger in early passage fibroblasts. Our results show that the induction of NF-kappaB-mediated signaling by UVB light is highly attenuated in senescent fibroblasts. This attenuation may reduce the stress resistance during cellular senescence.     

Effects of extracellular calcium on the subcellular translocation of bovine parathyroid PKC isozymes

The release of parathyroid hormone is regulated by the extracellular concentration of Ca2+ through a sensor(s) on the surface of the parathyroid cells, but few details are known on the further relay of the signal inside the cell. Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell soluble fraction to the particulate fraction of the cell. Therefore, identification of a subcellular localization of a PKC isozyme in parathyroid cells as a response to changes in extracellular Ca2+ should be an indication for its putative role in signal transduction coupled to the Ca2+ sensor. We have determined the subcellular localization of six PKC isozymes (alpha, betaI, betaII, epsilon, zeta, and iota) in nonstimulated parathyroid cells and in those treated with low (0.5 mM) and high (3.0 mM) extracellular Ca2+ by confocal microscopy. At the physiological concentration of serum Ca2+, all PKC isozymes studied were localized mainly to the cytosol, although to different extents. Low extracellular Ca2+ caused a redistribution of PKCalpha to the periphery of the cells. In contrast, PKCbetaI, -epsilon, -zeta, and -iota were translocated to the periphery of the cells at high extracellular Ca2+. These results indicate that PKCalpha, -betaI, -epsilon, -zeta, and -iota are involved in the response of parathyroid cells to changes in extracellular Ca2+.     

Identification of a hydrogen bond in the phe M197-->Tyr mutant reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides by X-ray crystallography and FTIR spectroscopy

In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X-ray structure analysis of the Phe M197-->Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 A resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al. [Biochemistry 32, 12875-12886, 1993] between the Tyr M197 hydroxy group and one of the 2a-acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the pi-conjugated 9-keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.     

Physiological consequence of disruption of the VMA1 gene in the riboflavin overproducer Ashbya gossypii

The vacuolar ATPase subunit A structural gene VMA1 of the biotechnologically important riboflavin overproducer Ashbya gossypii was cloned and disrupted to prevent riboflavin retention in the vacuolar compartment and to redirect the riboflavin flux into the medium. Cloning was achieved by polymerase chain reaction using oligonucleotide primers derived form conserved sequences of the Vma1 proteins from yeast and filamentous fungi. The deduced polypeptide comprises 617 amino acids with a calculated molecular mass of 67.8 kDa. The deduced amino acid sequence is highly similar to that of the catalytic subunits of Saccharomyces cerevisiae (67 kDa), Candida tropicalis (67 kDa), and Neurospora crassa (67 kDa) with 89, 87, and 60% identity, respectively, and shows about 25% identity to the beta-subunit of the FoF1-ATPase of S. cerevisiae and Schizosaccharomyces pombe. In contrast to S. cerevisiae, however, where disruption of the VMA1 gene was conditionally lethal, and to N. crassa, where viable disruptants could not be isolated, disruption of the VMA1 gene in A. gossypii did not cause a lethal phenotype. Disruption of the AgVMA1 gene led to complete excretion of riboflavin into the medium instead of retention in the vacuolar compartment, as observed in the wild type.