Inhibition of fungal growth by plant chitinases andβ-1,3-glucanases

Summary Plant chitinases and -1,3-glucanases have been demonstrated to inhibit fungal growth in model experiments, both on agar plates or in liquid media. Here,Trichoderma longibrachiatum was taken as a model to study the morphological changes caused by chitinase and glucanase treatments, using cytochemical techniques in combination with fluorescence and electron microscopy. Chitinase, alone or in the presence of glucanase, arrested growth of the hypha: it affected the extreme tip of the fungus producing a thinning of the wall, a balloon-like swelling and a rupture of the plasma membrane. Chitin and glucans were present in the wall, as shown by lectinand enzyme-binding experiments, but they had a different susceptibility to chitinase and -1,3-glucanase. Chitin was present at the apex and in the inner parts of the lateral walls; it was more susceptible to chitinase at the tip than in the subapical part. Glucans mostly occurred on the outer layer where they were degraded by glucanase. The latter did not affect the inner hyphal skeleton. It is suggested that the growth inhibition ofTrichoderma by hydrolytic enzymes is the consequence of a thinning of the cell wall in the hyphal apex, leading to an imbalance of turgor pressure and wall tension which causes the tip to swell and to burst.Abbreviations WGA-FITC wheat germ agglutinin labelled with fluorescein isothiocyanate - ConA-FITC concanavalin A labelled with fluorescein isothiocyanate - PEG polyethylene glycol - SEM scanning electron microscopy - TEM transmission electron microscopy     

Calibration of a resonance energy transfer imaging system.

A quantitative technique for the nondestructive visualization of nanometer scale intermolecular separations in a living system is described. A calibration procedure for the acquisition and analysis of resonance energy transfer (RET) image data is outlined. The factors limiting RET imaging of biological samples are discussed. Measurements required for the calibration include: (a) the spectral sensitivity of the image intensifier (or camera); (b) the transmission spectra of the emission filters; and (c) the quantum distribution functions of the energy transfer pair measured in situ. Resonance energy transfer imaging is demonstrated for two DNA specific dyes. The Förster critical distance for energy transfer between Hoechst 33342 (HO) and acridine orange (AO) is 4.5 +/- 0.7 nm. This distance is slightly greater than the distance of a single turn of the DNA helix (3.5 nm or approximately 10 base pairs), and is well below the optical diffraction limit. Timed sequences of intracellular energy transfer reveal nuclear structure, strikingly similar to that observed with confocal and electron microscopy, and may show the spatial distribution of eu- and hetero- chromatin in the interphase nuclei.     

Staphylococcus muscae, a new species isolated from flies.

A new coagulase-negative species of the genus Staphylococcus, Staphylococcus muscae, is described on the basis of the results of a study of four strains that were isolated from flies. 16S rRNA sequences of the type strains of S. muscae, Staphylococcus schleiferi, and Staphylococcus sciuri were determined and used, together with the corresponding sequences of Staphylococcus aureus and Staphylococcus epidermidis, for a comparative analysis. The new species is characterized taxonomically; this species is differentiated from the other novobiocin-susceptible staphylococci by its physiological and biochemical activities, cell wall composition, and levels of genetic relatedness. The type strain of this species is strain MB4 (= CCM 4175).     

Paracoccus denitrificans cytochrome c1 gene replacement mutants.

We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria.     

Formation of lipid-bound oligosaccharides in yeast

Incubation of a membrane fraction from Saccharomyces cerevisiae with UDP-N-acetyl [¹⁴C] glucosamine catalyzes the tranfer of N-acetylglucosamine to an endeenous lipid fraction as well as a methanol-insoluble polymer. The glycolipid was shown to separate into three compounds by thin-layer chromatography. The biosynthesis of two of them could clearly be stimulated by the addition of dolichol monophosphate to the incubation mixture. Evidence is presented that the substances are dolichol pyrophosphate derivatives: dolichol pyrophosphate N-acetylglucosamine and dolichol pyrophosphate di-N-acetylchitobiose. The formation of the chitobiose-containing lipid was increased by reincubation of the glycolipid with non-radioactive UDP-N-acetylglucosamine.The same particulate preparation transferred mannose from GDPmannose to dolichol pyrophosphate di-N-acetylchitobiose, giving rise to a lipid-bound oligosaccharide. Molecular weight determination of the oligosaccharide moiety gave a value of 780, which is consistent with a tetrasaccharide containing two mannose subunits attached to di-N-acetylchitobiose.The methanol-insoluble radioactive product obtained in the presence of UDP-N-acetyl[¹⁴C]glucosamine was transformed by pronase treatment to a large extent into dialyzable material. It is suggested that the glycolipids described serve as intermediates in the glycosylation of yeast mannoproteins.     

Elektronenmikroskopische Untersuchungen anPaulinella chromatophora Lauterborn,einer Thekamöbe mit blau-grünen Endosymbionten (Cyanellen)

Zusammenfassung Die Feinstruktur der Cyanellen vonPaulinella chromatophora sowie die Bildung und Struktur der Kieselschuppen, die das Gehäuse dieser Thekamöbe aufbauen, wurden untersucht.Die beiden wurstförmigen Cyanellen besitzen eine 6–13 nm dicke Wandschicht. Sie liegen eingeschlossen in Vesikeln im Cytoplasma des Wirtes. Das Chromatoplasma der Cyanelle enthält 15–20 konzentrisch angeordnete Thylakoide, Plastoglobuli und Phycobilisomen. Das Centroplasma enthält polyedrische Körper.Das Gehäuse der Thekamöbe besteht aus verkieselten rechteckigen Schuppen, die sehr regelmäßig zum Gehäuse zusammengefügt sind. Die Schuppen haben eine komplizierte Feinstruktur. Sie entstehen, vielleicht unter Mitwirkung von Mikrotubuli, vor der Zellteilung in Vesikeln, die wahrscheinlich aus Zisternen des einzigen Dictyosomes der Thekamöbe hervorgehen. Dieses Dictyosom liegt dem Zellkern am aboralen Pol derPaulinella an. Hexagonale Körper (Virionen?) werden aus dem Zellkern und dem Cytoplasma des Wirtes beschrieben.

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Electron microscopical investigations onPaulinella chromatophora Lauterborn, a thecamoeba containing blue-green endosymbionts (cyanelles)

Summary The ultrastructure of the sausage-shaped cyanelles and the ultrastructure and formation of the thecal scales ofPaulinella chromatophora were investigated. The cyanelles have a 6–13 nm thick wall. They are lying within vesicles in the cytoplasma of the host. The chromatoplasma has 15–20 concentrically arranged thylakoids, plastoglobuli and phycobilisomes. The centroplasma contains polyhedral bodies. The theca ofPaulinella chromatophora is composed of rectangular scales arranged in a very regular manner. These scales exhibit a very complex ultrastructure. They are produced prior to cell division in large vesicles probably derived from cisternae of the only dictyosom which is located close to the nucleus in the aboral part of the thecamoeba. Microtubules may play a role in the morphogenesis of these scales.Hexagonal particles (virions?) are described from the nucleus and the cytoplasma of some of the thecamoebae.

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FräuleinBrigitte Schendel danke ich für ihre gute technische Mitarbeit, der Deutschen Forschungsgemeinschaft danke ich für Sachbeihilfe.     

Serum ferritin and the iron status of Canadians.

Serum ferritin concentration was determined in 1105 Canadians aged 1 to 90 years. Geometric mean values (ng/ml) were as follows: children 1 to 4 years old, 12; children 5 to 9 years old, 15; adolescent girls, 17; adolescent boys, 18; women 20 to 39 years, 23; women 65 years and older, 52; men 20 to 39 years, 93; and men 40 and older, 92. Ranges were side in all age groups, reflecting variations in size of body iron stores. From analysis of the ferritin values it is highly probably that iron stores were greatly reduced in approximately 25% of children, 30% of adolescents, 30% of menstruating women, 60% of pregnant women and 3% of men. Iron-deficiency anemia was noted in only 2% of subjects. If "normality" requires more than small amounts of storage iron to meet physiologic demands, the study results suggest a high probability of iron deficiency in 60% of the pregnant women and in 19% of the other subjects; but if normality is defined as maintenance of adequate iron stores for erythropoiesis, the prevalence of iron deficiency was zero in the pregnant women and 2% in the other subjects.     

DNA is present in the nucleomorph of cryptomonads: Further evidence that the chloroplast evolved from a eukaryotic endosymbiont

Summary The nucleomorph is a unique self-replicating organelle which is invariably present in the periplastidal compartment of cryptomonads. The nucleomorph ofCryptomonas abbreviata is located in a groove on the inner face of the pyrenoid. When JB-4-embedded sections ofC. abbreviata are stained with 4-6-diamidino-2-phenylindole (DAPI), the nucleomorph exhibits a blue fluorescence characteristic of DNA-DAPI complexes. This fluorescence is removed by DNase digestion, but not by RNase. When cells are prepared for electron microscopy by the method of Ryter and Kellenberger (Schreil 1964), a network of fine DNA-like fibrils is observed in the nucleomorph matrix. It is estimated that the nucleomorph contains between 10⁸ and 10⁹ daltons of DNA. The presence of DNA in nucleomorphs strongly supports the hypothesis that the nucleomorph is the vestigial nucleus of a eukaryotic endosymbiont. It is postulated that this eukaryotic symbiont was an ancestral red alga or an organism closely related to red algae. The cryptomonad host cell, on the other hand, is not evolutionarily close to any other group of algae.     

Acute leukemia with chromosome translocation (4;11): 7 new patients and analysis of 71 cases

Clinical and laboratory features of seven patients with acute leukemia associated with the (4;11) chromosome translocation are presented. Leukemic blasts of these patients showed lymphoid morphology in 6 (although 1 was treated for monoblastic leukemia 3 years earlier) and monocytoid morphology in 1, were positive for TdT and HD 37 (CD 19) in 6 patients, whereas weak expression of CALLA was seen in only 1 patient and T-lineage-associated antigens in none. Leukemic blasts from four patients showed the simultaneous expression of B-lymphoid and myeloid antigens, suggesting leukemogenesis in a very early multipotent progenitor cell. In 2 patients an isochromosome of the long arm of No. 7 chromosome was found in the leukemic karyotypes in addition to t (4; 11) (q 21; q 23); in one instance present at diagnosis, in the other one occurring at relapse. In one other patient leukemia karyotype also demonstrated trisomy 8. Leukemic cells of three patients were investigated by molecular genetics and demonstrated immunoglobulin gene rearrangements for the Ig heavy chain sequences but not for the light chain constant regions and T cell receptor sequences. All patients were treated by intensive chemotherapy. Four of the 7 patients are in continuous complete remission. The longest event-free survival time (over 2 1/2 years) was seen in one patient who had also DOWN-syndrome. Including these 7 patients a clinical analysis of 71 patients with t (4; 11) acute leukemia was made, emphasizing the following characteristics at diagnosis: female sex (62%), age under 2 years (49%), leukocyte count over 100 X 10(9)/1 (61%), splenomegaly (80%), CNS-disease (11%). Survival of over 2 years was reported in less than 15% of the patients. It remains to be seen if risk-adapted treatment can alter the course of this early B-precursor acute leukemia with hitherto very bad prognosis.     

The phylogenetic position of Streptococcus and Enterococcus

Streptococcus pyogenes, S. equinus, S. bovis, S. salivarius, S. sanguis, S. mutans, S. rattus, S. cricetus, S. lactis, S. raffinolactis and Enterococcus faecalis have been characterized by oligonucleotide cataloguing of their 16S ribosomal RNA. All the organisms form a loose but coherent group that is phylogenetically equivalent to those of lactobacilli, bacilli, the Brochothrix and Listeria group, and related taxa that constitute one of several sublines within the 'Clostridium' branch of Gram-positive eubacteria. Within the Steptococcus-Enterococcus group, organisms fall into three moderately related clusters defined by Enterococcus, the lactic acid streptococci and streptococci of the pyogenic and oral groups, respectively.