Binding of β4γ5 by adenosine A1 and A2A receptors determined by stable isotope labeling with amino acids in cell culture and mass spectrometry

Characterization of G protein βγ dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity, or availability. As a new approach, we used quantitative mass spectrometry to characterize native βγ dimers associated with adenosine A(1):α(i1) and adenosine A(2A):α(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):α(i1) were cultured in media containing [(13)C(6)]Arg and [(13)C(6)]Lys and βγ labeled with heavy isotopes purified. Heavy βγ was combined with either recombinant βγ purified from Sf9 cells, βγ purified from the A(2A):α(S) expressed in HEK-293 cells cultured in standard media, or an enriched βγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, protein bands containing β and γ were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three β isoforms, β(1), β(2), and β(4), and seven γ isoforms, γ(2), γ(4), γ(5), γ(7), γ(10), γ(11), and γ(12), were identified in the analysis. β(1) and γ(5) were most abundant in the enriched βγ fraction, and this βγ profile was generally mirrored in the fusion proteins. However, both A(2A):α(S) and A(1):α(i1) bound more β(4) and γ(5) compared to the enriched βγ fraction; also, more β(4) was associated with A(2A):α(S) than A(1):α(i1). Both fusion proteins also contained less γ(2), γ(10), and γ(12) than the enriched βγ fraction. These results suggest that preferences for particular βγ isoforms may be driven in part by structural motifs common to adenosine receptor family members.     

Subependymale Basalmembranlabyrinthe im Hinterhorn des Seitenventrikels des Kaninchengehirns

Zusammenfassung Die Wand des Hinterhorns (Seitenventrikel des Kaninchengehirns), begrenzt von Radiatio corporis callosi und Alveus, ist von einem einschichtigen kubischen Ependym bedeckt. Unter diesem liegen zahlreiche weite Kapillaren und Venen, umgeben von zwei Basalmembranen, die einen perivaskulären Spalt einschließen, während die äußere Basalmembran ein Labyrinth von verzweigten Ausstülpungen bildet. Dieses Labyrinth erstreckt sich bis zu einer Entfernung von etwa 1 m gegen den Ventrikel. Die funktionelle Bedeutung der Strukturen wird diskutiert.

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Subependymal basement labyrinths in the posterior horn of the lateral ventricle of brain in rabbitThe question of liquor drainage

Summary The wall of the posterior horn of the lateral ventricle of brain (rabbit), lined by the Radiatio corporis callosi and the Alveus, is covered with a simple cuboidal ependyma. The underlying tissue contains numerous large capillaries and veins, which are surrounded by two basement membranes enclosing a perivascular space and forming a labyrinth of ramified extrusions of the outer basement membrane. This labyrinth is directed at the ventricle up to a distance of 1 m. The functional significance of these structures is discussed.

Die Untersuchung wurde mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft durchgeführt.     

Synapsenartige Kontakte zwischen intraventrikulären Axonendigungen und freien Oberflächen von Ependymzellen des Kaninchengehirns

Zusammenfassung Im III. und IV. Ventrikel des Kaninchengehirns kommen zahlreiche nackte Axone mit kolbenförmigen Endigungen vor, die kleine helle Vesikel (400–800 Å), Vesikel mit dichtem Kern (650–1000 Å) und Mitochondrien mit einem prismatischen Tubulus enthalten. Das Plasmalemm der Kolben bildet mit dem apikalen Plasmalemm des Ependyms synapsenähnliche Kontakte. Sie sind durch praesynaptische dense bodies, Membranverdickung, subjunctional bodies und parallele Filamente (50 Å) charakterisiert, die den synaptischen Spalt (200 Å) überqueren. Außerdem kommen zwischen den Kolben und dem Ependym desmosomenähnliche Kontakte vor.

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Synapse-like contacts between the intraventricular axonal endbulbs and the apical plasmalemma of ependyma (rabbit)

Summary In the 3rd and 4th ventricle of the brain (rabbit) a great number of non-myelinated axons are found having bulb like endings. These terminals contain small clear vesicles (400–800 Å), dense cored vesicles (650–1000 Å), and mitochondria, wich are characterized by a single prismatic tubule. The plasmalemma of the bulb is in a synapse-like contact with the apical plasmalemma of the ependyma. The contact is characterized by presynaptic dense bodies, membrane thickening, subjunctional bodies, and parallel intersynaptic filaments (50 Å) across the synaptic gap (200 Å). Besides, between the bulbs and ependyma desmosome-like junctions are found.

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Die Untersuchung wurde mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft durchgeführt.     

Plasmazellen in der Wand der Eminentia mediana des Kaninchens

Zusammenfassung In der Wand der Eminentia mediana des Kaninchens kommen Plasmazellen vor. Jede von ihnen ist umgeben von einem wechselnd weiten perizellulären Spalt, von markscheidenführenden und markscheidenfreien Axonen und von Glia- und Ependymfortsätzen. Eine Basalmembran zwischen Plasmazellen und Ependym fehlt. Plasmazellen werden auch im perivaskulären Spalt und im Ventrikellumen des Recessus infundibuli gefunden.

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Plasma cells in the wall of the median eminence in rabbit

Summary In the wall of the median eminence (rabbit) plasma cells occur. Each of them is surrounded by a variable pericellular space, by nonmyelinated and myelinated axons and by glial and ependymal processes. A basement membrane between the ependyma and the plasma cells does not exist. Plasma cells also are to be found in the perivascular space and in the cavity of the recessus infundibuli.

Die Untersuchung wurde mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft durchgeführt.     

Cytosomen mit Zylindroiden und fünfschichtigen Membranen. Untersuchungen an den Nerven- und Gliazellen der Area postrema im Kaninchengehirn

Zusammenfassung Die Area postrema vom Kaninchen wurde nach Fixierung durch Perfusion elektronenmikroskopisch untersucht.Nervenzellen können auf Grund von Strukturmerkmalen von Gliazellen unterschieden werden. Die Nervenzellen besitzen axo-somatische und axo-dendritische Synapsen von einfachem Bau. In beiden Zelltypen kommen Cytosomen hauptsächlich in der Golgi-Region vor. Die Cytosomen der Gliazellen enthalten eine granuläre Matrix, in der Membranen und manchmal lipidartige Tröpfchen vorkommen. Die Cytosomen der Nervenzellen enthalten granuläre Matrix, Membranen und charakteristische Zylindroide. Bis zu 40 Zylindroide werden innerhalb eines Anschnittes von Cytosomen gefunden. Die Zylindroide sind etwa 0,1–0,78 m lang, 0,05–0,175 m dick. Ihr Mantel besteht aus 2–7fach gewickelten Membranen, die einen zentralen Innenraum von 100–450 (-750) Å Durchmesser umgeben. Die intracytosomalen Membranen stellen einen eigenen Membrantyp dar. Sie sind fünfschichtig im Gegensatz zur Cytosomenhüllmembran, die mit der verwendeten Fixierung dreischichtig erscheint. Die Bedeutung der Cytosomen, ihrer fünfschichtigen Membranen und der für die Area postrema charakteristischen Zylindroide werden diskutiert im Hinblick auf ihre Ähnlichkeit mit Lysosomen und Mastzellgranula. Es wird darauf hingewiesen, daß Monoamine sowohl in Mastzellen als auch in Nervenzellen der Area postrema nachgewiesen worden sind.

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Summary The area postrema of rabbits was fixed by perfusion and investigated by electron microscopy.Nerve cells can be distinguished from glial cells by structural properties. Axo-somatic and axo-dendritic synapses of a simple type are found at the nerve cells.In both cell types, cytosomes are found mainly in the Golgi region. The cytosomes of glial cells are filled with a granular matrix, in which membranes and sometimes lipid-like droplets may be observed. The cytosomes of nerve cells contain a granular matrix, membranes and characteristic cylindroids. Up to 40 cylindroids are observed within one profile of cytosomes. The cylindroids are about 0.1–0.78 m long, 0.05–0.175 m thick. Their wall is built by 2–7 wrapped membranes, which surround the inner less dense core of 100–450 (-750) Å diameter. The cytosomal membranes represent a peculiar type of membranes, since they are of the five layered type, in contrast to the limiting membrane of the cytosomes which is a three layered membran under the conditions of fixation used. The significance of the intracytosomal five layered membranes, the cytosomes of glial and nerve cells and the cylindroids is discussed with respect to their similarity with lysosomes and mast cell granules. Attention is drawn to the observations of monoamines by other authors in mast cells as well as in nerve cells of the area postrema of the rabbit.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Surface morphology of the subfornical organ in the rabbit's brain

Summary The ventricular surface of the subfornical organ of the rabbit's brain was studied with scanning and transmission electron microscopic techniques. The ependymal covering was found to consist of hexagonal cells with convex apical surfaces. From the center of each cellular surface a single kinocilium up to 6 m in length protrudes into the liquor. It is usually covered with secretory material having the shape of pearlstrings. The surface aspect of the subfornical organ suggests secretion into the liquor by emptying of giant vacuoles which originate below the ependyma in nerve cells, move towards the surface, develop pressure while flattening their ependymal cover and finally erupt, leaving collapsed ependyma- and/or nerve cells bag on the surface of the organ. A second mechanism of more granular secretion by ependymal cells appears possible.We are indebted to Fräulein E. Östermann, Frau L. Schulze and Frau H. Zuther-Witzsch for excellent technical assistance.     

Characterization of the Mus308 Gene in Drosophila Melanogaster

Among the available mutagen-sensitive mutations in Drosophila, those at the mus308 locus are unique in conferring hypersensitivity to DNA cross-linking agents but not to monofunctional agents. Those mutations are also associated with an elevated frequency of chromosomal aberrations, altered DNA metabolism and the modification of a deoxyribonuclease. This spectrum of phenotypes is shared with selected mammalian mutations including Fanconi anemia in humans. In anticipation of the molecular characterization of the mus308 gene, it has been localized cytogenetically to 87C9-87D1,2 on the right arm of chromosome three. Nine new mutant alleles of the gene have been generated by X-ray mutagenesis and one was recovered following hybrid dysgenesis. Characterization of these new alleles has uncovered additional phenotypes of mutations at this locus. Homozygous mus308 flies that have survived moderate mutagen treatment exhibit an altered wing position that is correlated with reduced flight ability and an altered mitochondrial morphology. In addition, observations of elevated embryo mortality are potentially explained by an aberrant distribution of nuclear material in early embryos which is similar to that seen in the mutant giant nuclei.     

The C-terminal region of Ge-1 presents conserved structural features required for P-body localization

The removal of the 5′ cap structure by the DCP1–DCP2 decapping complex irreversibly commits eukaryotic mRNAs to degradation. In human cells, the interaction between DCP1 and DCP2 is bridged by the Ge-1 protein. Ge-1 contains an N-terminal WD40-repeat domain connected by a low-complexity region to a conserved C-terminal domain. It was reported that the C-terminal domain interacts with DCP2 and mediates Ge-1 oligomerization and P-body localization. To understand the molecular basis for these functions, we determined the three-dimensional crystal structure of the most conserved region of the Drosophila melanogaster Ge-1 C-terminal domain. The region adopts an all α-helical fold related to ARM- and HEAT-repeat proteins. Using structure-based mutants we identified an invariant surface residue affecting P-body localization. The conservation of critical surface and structural residues suggests that the C-terminal region adopts a similar fold with conserved functions in all members of the Ge-1 protein family.     

Protein targeting to subnuclear higher order structures: A new level of regulation and coordination of nuclear processes

Though there are no separating membranes within the nucleus, different factors are often concentrated at sites where their respective function is required, a phenomenum referred to as functional organization of the nucleus. How is then this organization achieved and how are the different metabolic processes integrated in the nucleus? One emerging principle was revealed by the identification of protein domains that, though not involved in catalysis, regulate enzyme activity at a higher order level by targeting enzymes to the right place at the right time. These targeting sequences constitute an assembly code for nuclear ‘protein factories,’ which ensure the extremely high efficiency and accuracy needed in a complex and competitive environment as the living mammalian cell. J. Cell. Biochem. 70:222– 230, 1998. © 1998 Wiley-Liss, Inc.