Radioimmunoassay of cyclic AMP can provide a highly sensitive assay for adenylate cyclase, even at very high ATP concentrations

Modifications of the cyclic AMP radioimmunoassay of Cailla et al. [in Hormones and Cell Regulation (J. Dumont and J. Nunez, eds.), Vol. 4, pp. 1-24, Elsevier/North-Holland, Amsterdam/New York (1980)] allowed its use in the determination of adenylate cyclase activity, which was otherwise precluded by high blank values. These high values originate mainly from chemically formed cyclic AMP and from ATP cross-reactivity. The simultaneous presence of ATP and magnesium ions generates cyclic AMP under the alkaline conditions used to succinylate the sample; this interference can be dealt with either by chelation of Mg2+ ions with EDTA during succinylation or by periodic acid oxidation of samples prior to succinylation. In addition, ATP itself contributes to blank values by its cross-reactivity, especially when working with high concentrations of this substrate. This interference can be decreased by a batch adsorption of ATP or oxidized ATP on alumina. Detailed procedures were discussed, with the choice of the additional steps to the standard method of Cailla et al. having to be made on the basis of the sensitivity requirements. When preventing ATP cyclization, the radioimmunoassay was as sensitive as methods using [alpha-32P]ATP as substrate. Elimination of ATP can improve the sensitivity by one order of magnitude. This method is especially interesting with high ATP concentrations and/or with low cyclic AMP production.     

The fine structure of protonephridia in Gnathostomulida and their comparison within Bilateria

Summary The fine structure of the protonephridia of Haplognathia rosea (Filospermoidea) and Gnathostomula paradoxa (Bursovaginoidea) is described. Each protonephridium consists of three different cells: (1) a monociliated terminal cell which constitutes the filtration area, (2) a nonciliated canal cell showing a special protonephridial outlet system, and (3) an intraepidermal cell — the nephroporus cell — constituting the nephroporus. The protonephridia are arranged serially. There is no canal system connecting the protonephridial units.Protonephridial characters in other Bilateria are considered. The pattern of characters in the protonephridia in the last common gnathostomulid stem species and presumed apomorphies in the protonephridia of the Gnathostomulida investigated are discussed.Abbreviations used in figures ac acessory centriole - AC additional epidermal cell - bb basal body - bl basal lamina - bm bundle of microvilli - c cilium - cc cilium duct cell - cd cilium duct - cr ciliary rootlet - crs structures resembling ciliary rootlets - di diplosome - ds desmosome - dy dictyosome - f filtration area - g granules - m mitochondrium - mv microvillus - n nucleus - NC nephroporus cell - np nephroporus - oc outlet canal - TC terminal cell - tl tubules of lacunar system     

Isolation and structural analysis of two lipid A precursors from a KDO deficient mutant of Salmonella typhimurium differing in their hexadecanoic acid content

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-d-manno-octulosonic acid region of lipopolysaccharides.Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue.The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-d-manno-octulosonic acid and/or other polar substituents.Abbreviations LDMS laser desorption mass spectrometry - KDO 3-Deoxy-d-manno-octulosonic acid - Ts5 Salmonella typhimurium mutant Ts5 - PCP phenol/chloroform/petroleum ether - H₂F₂ hydrogen fluoride This work is dedicated to Prof. Dr. Drews, Freiburg, on the occasion of his 60th birthday     

Über bau und funktion der prosoma-drüsen von spinnmilben (Tetranychidae,Trombidiformes)

Zoomorphology - Die licht- und elektronenmikroskopische Untersuchung der Prosomadrüsen der Spinnmilben Bryobia praetiosa, Bryobia rubrioculus und Tetranychus urticae (Tetranychidae,...     

Über Aufbau und Innervation der Kopfanhänge der prosobranchen Schnecken

Zusammenfassung Das Epithel der Kopfanhänge von elf marinen und Süßwasserprosobranchiern besteht aus prismatischen bis kubischen Stützzellen mit meist dichtem Mikrovillussaum und z.T. Pigmentgranula sowie Sinneszellen, die fast immer in Form sekundärer Sinneszellen vorliegen; nur bei Patella coerulea kommen vermutlich auch primäre Sinneszellen vor. Ihr Zytoplasma ist apikal durch glattwandige E. R.-Zisternen, helle Bläschen und Mikrotubuli gekennzeichnet. Außerdem tragen diese Zellen Zilien und stehen basal mit Nervenendigungen in Kontakt, die sich in drei Gruppen einteilen lassen: 1. Vermutlich cholinerge Endigungen mit optisch leeren Bläschen (Ø 600–800 Å). 2. Endigungen mit dense core vesicles (Ø 1000–1100 Å). Die Annahme, daß diese Endigungen biogene Amine enthalten, wird durch fluoreszenzmikroskopische Befunde gestützt. 3. Endigungen mit großen (Ø 3000–4000 Å) neurosekretorischen Elementargranula.

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Structure and innervation of the cephalic tentacles of Prosobranch molluscs

Summary The epithelium of the cephalic tentacles of eleven marine and freshwater prosobranch snails consists of villus bearing supporting cells, which partly contain pigment granules, and sensory cells, which occur in form of secondary sensory cells with the exception of Patella coerulea which presumably possesses primary sensory cells. These receptor cells are characterized as chemoreceptors by apical cilia, smooth surfaced E.R., microtubulues and empty vesicles. At their bases they are in close contact with nerve endings which can be classified in three groups: 1. presumably cholinergic endings with clear vesicles (Ø 600–800 Å). 2. endings with dense core vesicles (Ø 1000–1100 Å). The assumption that these endings contain biogenic amines is supported by positive fluorescence microscopical tests. 3. Endings with big (Ø 3000–4000 Å) neurosecretory elementary granules.

Herrn Prof. Dr. W. Bargmann danke ich für die Überlassung eines Arbeitsplatzes im Anatomischen Institut Kiel.     

Comparative Study of Cultured Burkitt Tumor Cells by Immunofluorescence, Autoradiography, and Electron Microscopy

Cultured Burkitt cells were examined by immunofluorescence, autoradiography, and electron microscopy in an effort to identify the stainable cells with those harboring herpes-type virus particles. Immediately after a 2-hr pulse of (3)H-thymidine, from 30 to 60% of the cells revealed heavy nuclear labeling. In most cases the grains were evenly dispersed, but in about 3 to 5% the grains showed a focal distribution and occasionally they extended into the cytoplasm. Such nuclear foci were rarely seen at 8 hr after the pulse. When the analysis was restricted to preselected immunofluorescent cells, up to 80% showed label at 8 hr and cytoplasmic grains were prominent. To reduce cellular deoxyribonucleic acid (DNA) synthesis, cells were X-irradiated with 3,000 to 6,000 R, and the isotope pulse was applied 1, 4, or 7 days later. Whereas the total number of labeled cells decreased in roughly twofold steps at the respective intervals (from 40 to 10%), the incorporation of (3)H-thymidine into fluorescent cells was not affected by X irradiation. In each series, about 70% of the fluorescent cells contained label when they were examined at 24 and 48 hr after the pulse, whereas at 8 and 72 hr fewer were positive. At the earlier intervals, unlabeled fluorescent cells most likely represented cells which had completed viral DNA synthesis prior to the pulse; at the later intervals, unlabeled fluorescent cells were probably cells which commenced viral replication after the pulse. These data support the conclusion that the immunofluorescent cells are the ones which harbor virus, and also confirm the expectation that the virus is a DNA virus from a member of the herpes group. This conclusion was firmly established by sectioning and electron microscopic examination of individual fluorescent cells, all of which contained numerous virus particles, whereas the nonstained cells prepared in a similar manner were free of them.     

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.     

Modification of tumor cells by a low dose of Newcastle disease virus

Summary Augmented tumor-specific T cell responses were observed against the high metastatic murine lymphoma variant ESb when using as immunogen ESb tumor cells that had been modified by infection with a low dose of Newcastle disease virus (NDV). Such virus-modified inactivated tumor cells (ESb-NDV) were potent tumor vaccines when applied postoperatively for active specific immunotherapy of ESb metastases. We demonstrate here that immune spleen cells from mice immunized with ESb-NDV contain enhanced immune capacity in both the CD4⁺, CD8^– and the CD4^–, CD8⁺ T cell compartments to mount a secondary-tumor-specific cytotoxic T cell response in comparison with immune cells from mice immunized with ESb. ESb-NDV immune CD4⁺, CD8^– helper T cells also produced more interleukin 2 after antigen stimulation than the corresponding ESb immune cells. There was no participation of either CD4⁺ or CD8⁺ virus-specific cells in the augmented response. The specificity of the T cells for the tumor-associated antigen remaind unchanged. Thus, there is the paradox that the virus-mediated augmentation of the tumor-specific T cell response in this system involves increased T helper activity but does not involve the recognition of viral epitopes as potential new helper determinants.Abbreviations CTL cytolytic T lymphocytes - IL-2 interleukin 2 - rIL-2 recombinant IL-2 - mAb monoclonal antibody - NDV Newcastle disease virus - SSC syngeneic spleen cell