Using protein synthesis inhibitors to establish the phylogenetic relationships of the sulfolobales order

The sensitivity of the cell-free protein synthesis systems from Acidanus brierleyi, Acidianus infernus, and Metallosphaera sedula, members of the archaeal order Sulfolobales, to 40 antibiotics with different specificities has been studied. The sensitivity patterns were compared to those of Sulfolobus solfataricus and other archaeal, bacterial, and eukaryotic systems. The comparative analysis shows that ribosomes from the sulfolobales are the most refractory to inhibitors of protein synthesis described so far. The sensitivity results have been used to ascertain in phylogenetic relationships among the members of the order Sulfolobales. The evolutionary significance of these results are analyzed in the context of the phylogenetic position of this group of extreme thermophilic microorganisms. Correspondence to: R. Amils     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

Amino acid pool and protein turnover during differentiation (spherulation) ofPhysarum polycephalum

The composition of the amino acid pool during spherulation was determined. It changes in size and in composition, the concentration of each amino acid behaving individually. The first response to the onset of spherulation either by starvation or osmotic shock (0.5 M mannitol) always is a decrease of the pool's size, which during further starvation expands for a short period and then decreases again. During development induces by mannitol in the presence of external amino acids, the pool size increases continuously after the initial depletion.As shown by radioactive labeling, amino acids were actively released from the plasmodium into a medium containing amino acids, but retained by the microplasmodia in an amino acid-free medium. The kinetics of the uptake of radioactive amino acids from the medium is biphasic, indicating the existence of multiple pools. Even after a labeling period of 8 h the amino acid pool is not yet in equilibrium with the medium. The possibility of a compartimentation of the pool was confirmed by density labeling of two different enzymes.Whereas the turnover of total protein is only very low during growth, it is rather high in spherulating microplasmodia. At least 70% of the originally existing protein is degraded during this development, while, simultaneously, at least 50% of the protein present after 24 h starvation is newly synthesized during that period.     

Effect of ultraviolet radiation on the microsporidian Octosporea muscaedomesticae with reference to protectants provided by the host Phormia regina

The effect of ultraviolet light on the microsporidian Octosporea muscaedomesticae in relation to infection in the adult black blowfly, Phormia regina, was investigated. A 30-Watt germicidal lamp, 253.7-nm wavelength, was used as source of uv light in five investigations. In addition, sunlight served as a uv source in two studies. Viable naked dried spores exposed to the uv lamp at a distance of 10 cm were killed after 15 min. Viable naked spores in an aqueous suspension were killed after 30 min of exposure to the uv lamp and after 3 hr of exposure to bright sunlight, respectively. Daily 30-min uv lamp exposures on living hosts harboring all life phases of the parasite did not interfere with the ensuing infection in the blowfly's midgut and the pathogen's developmental cycle. Spores harvested from uv-treated infected hosts were found to be as infective as spores retrieved from hosts not treated with uv. Spores contained in dried fecal droplets and exposed up to 3 hr to the uv lamp, or 12 hr to bright sunlight, respectively, remained infective. Addition of uric acid to a preparation of naked spores prior to 15- and 30-min uv irradiations yielded 100% infection in both host groups. A uv-protective function is ascribed to components provided by the host's tissues and feces.     

Ergänzende Untersuchungen zur Ultrastruktur von Trichoplax adhaerens F.E. Schulze (Placozoa)

Zusammenfassung 1. Die Zellen des Ventralepithels bilden keine Mikrovilli, sondern ein schwammartiges Maschenwerk gefensterter Leisten und Falten, welches bei der Haftung des Tieres an der Unterlage und/oder bei der extrasomatischen Vorverdauung der Nahrung eine Rolle spielen dürfte.2. Während der Zellteilungen treten typische Centriole an den Spindelpolen auf.3. Die wachsenden Eizellen phagocytieren Fortsätze der Faserzellen, die als Trophocyten fungieren.4. Unter den gleichen Bedingungen, die zur Eibildung führen, können sogen. S-Zellen auftreten. Einige ultrastrukturelle Befunde sprechen dafür, daß es sich um Spermien handelt.

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Additional investigations on the ultrastructure of Trichoplax adhaerens F.E. Schulze (Placozoa)

Summary 1. The cells of the ventral epithelium form no microvilli but a spongy meshwork of fenestrated ledges and folds which may play a rôle in the adhesion of the animal to the substratum and/or in the extrasomatic predigestion of the food. 2. During cell division typical centrioles occur at the spindle poles. 3. The growing egg cells phagocytize projections of the fiber cells which function as trophocytes. 4. Under the same conditions leading to egg formation so-called S-cells may occur. Some ultrastructural data suggest that they are sperm cells.

     

Cocultivation studies with cells of patients bearing fragile X chromosomes

Summary Fourteen cocultivation studies were carried out with cells of four patients with fragile X, one obligate and two possible female heterozygotes, two female controls, and a rabbit. In all cocultivations the number of fragile X chromosomes was sharply reduced in the patient cells. The strongest effect was caused by the animal cells. A distinct difference between the two controls in the reducing ability was observed. No such difference was found between the obligate and possible heterozygotes on the one hand and the controls on the other. To test the influence of the residual serum in the mixed blood cultures, the serum of a patient's blood sample was replaced by the serum of a control. The frequency of fragile X chromosomes was not decreased by this procedure. Therefore a soluble factor is supposed to exist which is produced by normal or heterozygote cells in culture and which reduces the expression of fragile sites in patient cells.     

Changes in the phytoplankton of Lake Trummen induced by restoration

Lake Trummen, previously an oligotrophic lake, was heavily polluted during 1930–1957. The lake was restored by suction dredging of the top sediment layer. After restoration nutrien concentrations and phytoplankton biomass were drastically reduced. Eutrophic species disappeared and more oligotrophic species returned to the lake. The investigation covers 12 years-monthly-bimonthly countings of algae together with electronmicroscopical studies of taxonomically difficult taxa. The investigation was made at the species level (300 species identified and 80 quantitatively recorded).     

Taxonomic relations between archaebacteria including 6 novel genera examined by cross hybridization of DNAs and 16S rRNAs

DNAs from 16 species of archaebacteria including 6 novel isolates were hybridized with 16S rRNAs from 7 species representing different orders or groups of the urkingdom of archaebacteria. The yields, normalized for the number of genes per microgram of DNA, and the temperature stabilities of all hybrids were determined and related to each other. A taxonomic tree constructed from such fractional stability data reveals the same major divisions as that derived from comparative cataloging of 16S rRNA sequences. The extreme halophiles appear however as a distinct order besides the three known divisions of methanogens. The methanogens, the halophiles and Thermoplasma form one of two clearly recognizable branches of the archaebacterial urkingdom. The order represented by Sulfolobus and the related novel order Thermoproteales form the other branch. Three novel genera, Thermoproteus, Desulfurococcus and the "stiff filaments" represent three families of this order. The extremely thermophilic methanogen Methanothermus fervidus belongs to the Methanobacteriales. SN1, a methanogen from Italy, appears as another species of the genus Methanococcus. Another novel methanogen, M3, represents a genus or family of the order Methanomicrobiales.     

Enzymatic ethylene formation from 1-aminocyclopropane-1-carboxylic acid by manganese,a protein fraction and a cofactor of etiolated pea shoots

The cofactor of enzymatic, 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation was concentrated on cation exchange columns. When chelators of cations were added to the homogenates, cofactor activity was lost. Cofactor fractions were partly resistant to oxidation at 600° C. Mn²⁺ substituted for the cofactor in ethylene formation from 1-aminocyclopropane-1-carboxylic acid by a protein fraction isolated from etiolated pea shoots. In addition, Mn²⁺ enhanced the stimulatory effect of the concentrated cofactor. The elution volume for the cofactor on a Sephadex G-25 column was lower than that of MnCl₂. In paper electrophoresis the cofactor migrated to the cathode at pH 10.8 and 2.2. The R_F of cofactor on cellulose plates developed in butanol: acetic acid: H₂O was 0.4. After cellulose chromatography, cofactor activity had to be reconstituted by the addition of MnCl₂. Chelators, anti-oxidants, and catalase were inhibitors of Mn²⁺-cofactor-dependent ethylene formation. The protein necessary for 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation in vitro was seperated from 95–98% of the total protein in homogenates by DE-52 cellulose chromatography and (NH₄)₂SO₄-fractionation.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EDTA ethylenediaminetetraacetic acid - DDTC diethyldithiocarbamate     

Hell-und Dunkeladaptation der Augen vonPieris brassicae L. (Lepidoptera)

Summary The compound eyes ofPieris brassicae L. have a tiered retina. During light and dark adaptation, ultrastructural changes have been observed throughout the length of the ommatidia in the latero-ventral region of the eyes. These changes have been quantitated by mapping at distinct levels of the ommatidia, and plotted as histograms. Both in visual cells and secondary pigment cells and at the attachment region between crystalline cone and rhabdome such ultrastructural changes have been found to be correlated to the state of adaptation.Distal and proximal photoreceptor cells show different adaptation mechanisms. Whereas the distal cells show a clear pupil mechanism in their distal parts, there is only very little horizontal movement of pigment granules in the proximal cells. In the proximal cells, multivesicular bodies (MVB) are always abundant, while in the distal cells their number is small and increases slightly during light adaptation. In the proximal cells light adaptation causes pigment granules, located in the distal process, to move proximally. Increasing the light intensity from 160 to 1600 W/cm² results in more intense migration of pigments.In the secondary pigment cells, a slight but significant distal movement of pigment granules is observed at high light intensity. If continued this condition causes the granules to aggregate in the vicinity of the apical cell membrane, and to move up to the distal inflated extensions of the distal processes formed by these cells. In dark adapted eyes, these processes are nearly devoid of pigment and the pigment granules beneath the apical membrane disperse. In addition to these structural changes, there is a tendency for retinal movements at the attachment from crystalline cone to rhabdome. — The various adaptation mechanisms are not equally well developed in different regions of the compound eye.

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Hell-und Dunkeladaptation der Augen vonPieris brassicae L. (Lepidoptera)

Zusammenfassung Die Retina vonPieris brassicae L. ist mehrreihig. Erstmals wurden feinstrukturelle Veränderungen während der Hell und Dunkeladaptation über die gesamte Länge der Ommatidien des latero-ventralen Augenbereichs anhand von Kartierungen in vergleichbaren Höhen der Ommatidien untersucht und in Histogrammen wiedergegeben. — Sowohl in den Sehzellen als auch Nebenpigmentzellen und am Übergang von Kristallkegel zum Rhabdom wurden feinstrukturelle Veränderungen in Korrelation mit der Adaptation gefunden.Die Adaptation erfolgt bei distalen und proximalen Sehzellen jeweils auf andere Art. Während die distalen Sehzellen in ihrem distalsten Bereich sehr gut die Pupillenreaktion zeigen, adaptieren die proximalen Sehzellen nur geringfügig mit horizontaler Pigmentwanderung. Auch die Anzahl der multivesikulären Körper (MVB), die in den proximalen Sehzellen immer groß ist, steigt bei Helladaptation (HA) nur in den distalen Sehzellen etwas an. In den proximalen Sehzellen wandern die Pigmentgranula bei HA geringfügig aus dem distalen Fortsatz dieser Sehzellen proximalwärts. Intensitätssteigerung auf das 10fache (von 160 auf 1600W/cm²) bewirkt eine Verstärkung der genannten Pigmentwanderungs-Reaktionen in den Sehzellen.Die Granula der Nebenpigmentzellen wandern bei HA mit starker Intensität etwas distalwärts. — Bei starker langer HA häufen sich diese Granula unter der apikalen Membran dieser Nebenpigmentzellen und wandern bis in die distalen kleinen Erweiterungen der distalen Fortsätze dieser Zellen. Bei Dunkeladaptation (DA) sind diese Fortsätze nahezu frei von Pigment; unter der apikalen Zellmembran verteilen sich die Pigmente locker. Außerdem besteht am Übergang von Kristallkegel zu Rhabdom die Tendenz zur Retinomotorik. — In den verschiedenen Augenbereichen erfolgen die genannten Adaptationsreaktionen unterschiedlich gut.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft und der Stiftung Volkswagenwerk

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Herrn Prof. Dr. Kurt Hamdorf (Bochum) danken wir für kritische Diskussion und Fräulein Althaus für die graphischen Darstellungen