Localization of chitin on ultrathin sections of cysts of two ciliated protozoa,Blepharisma undulans andPseudomicrothorax dubius,using colloidal gold conjugated wheat germ agglutinin

Summary The cyst walls of the ciliatesBlepharisma undulans andPseudomicrothorax dubius were examined ultrastructurally and by postembedding labeling with wheat germ agglutinin (WGA)-gold conjugate. Different methods of fixation and embedding were performed. In all procedures, WGA-gold binds selectively to material of the cyst wall. Pretreatment of the sections with chitinase inhibits labeling. The cyst walls of both species contain 3 nm fibrils, which are supposed to be of chitinous nature. In the cyst wall ofB. undulans, several thin layers of WGA-binding fibrils are interspaced with thick layers of other material. InP. dubius, WGA-binding sites are mainly concentrated in the mesocyst, where the microfibrils appear to represent the major component. These results obtained from two phylogenetically distant species confirm that chitin synthesis is an ancestral feature of ciliated protozoa. The amount and distribution of the chitin fibrils may play an important role in the properties and functions of the wall of the resting cyst.     

Amber Mutants of Bacteriophages T3 and T7 Defective in Phage-directed Deoxyribonucleic Acid Synthesis

Amber mutants of the related phages T3 and T7 were isolated and tested for their ability to restore-as the wild type does-thymidine incorporation in ultraviolet (UV)-irradiated, UV-sensitive, nonpermissive host bacteria (Escherichia coli B(s-1)). Most amber mutants had this ability. However, in both T3 and T7, mutants unable to promote thymidine incorporation under these conditions were found and classified into two well-defined complementation groups: T3DO-A and T3DO-B, T7DO-A and T7DO-B. Infection of B(s-1) cells with representatives of groups DO-A had the following characteristics: (i) phage-directed uridine uptake in UV-irradiated cells was reduced to less than 20% of normal; (ii) breakdown of host deoxyribonucleic acid (DNA) was delayed and incomplete; (iii) no serum-blocking antigens appeared; (iv) no cell lysis occurred; (v) the ability to exclude the heterologous wild type was impaired. Amber mutants of the DO-B groups, infecting B(s-1), were able to: (i) promote an efficient phage-directed uridine uptake in UV-irradiated cells; (ii) bring about rapid breakdown of host DNA; (iii) synthesize serum-blocking antigens; (iv) lyse the host cells, generally after the normal latent period; (v) exclude efficiently the heterologous wild type. Although physiological similarities between the respective DO-A mutants or DO-B mutants of T3 and T7 were evident, no physiological cross-complementation occurred, and genetic crosses gave no evidence of genetic homologies between groups of T3 and T7.     

Feinstrukturelle und mikroanalytische Untersuchungen an den Kristallen und Lithosomen des CiliatenEuplotes vannus

Summary In the cytoplasm of the marine ciliateEuplotes vannus, there exist two conspicuous types of membrane bound inclusions: 1. irregularly shaped crystals which are highly anisotropic; 2. globular lithosomes characterized by concentrically arranged layers of deposits which exhibit only faint birefringence. Normally, both structures form distinct accumulations. Energy dispersive X-ray microanalysis of these accumulations reveals a high content of calcium and phosphorus, besides magnesium, sulphur and chlorine. Analysis of cell areas devoid of the inclusions show significantly lower calcium- and phosphorus-peaks.

     

Background and peak evaluation of one parameter flow karyotypes on a PC/AT computer

Flow cytometry has become a fast, quantitative method for the classification of metaphase chromosomes in suspension (flow karyotyping) stained with fluorescent dyes. Such a flow karyotype (frequency distribution of the fluorescence signals) consists of several peaks. The peak pattern characterizes the analyzed chromosome complement. In many cases flow karyotypes contain a continuum of an unspecific background deriving from chromosome fragments or chromosome aggregates. For the quantitative evaluation of a flow karyotype this background has to be subtracted by a suitable background function. In this approach the application of chi 2-functions is described. The feasibility of this method to flow karyotypes has been concluded from a computer simulation of chromosome breaking under different conditions. In spite of the rather rough assumptions of the model compared to the complex reasons that influence chromosome breaking, the chi 2-function fits the background better than the exponential function in current use. The approximation of a Gaussian distribution function by the chi 2-function also makes it possible to use the same subtraction procedure for chromosome aggregates. The procedure was tested for isolated chromosomes of Chinese hamster cell lines under different states of breaking. For further evaluation of one parameter flow karyotypes a setup of computer routines has been developed for PC/AT and compatible computer systems. Different peak values of these flow karyotypes can be determined (e.g. peak mean, standard deviation, absolute and relative peak area etc.). The applied method is to fit Gaussian curves to each peak of an experimentally measured histogram by using an interactive program. Fluctuations depending on 'noise' may be suppressed by a 'k-nearest-neighbours' smoothing procedure.     

Zur Digestion bei Pseudomicrothorax dubius Mermod Nahrungsvakuolen-Vesikulation (Ciliophora) im Anschluß an die Phagocytose

Zusammenfassung Der Schlinger Pseudomicrothorax dubius ingestiert innerhalb von 1–2 min ein großes Volumen fädiger Blaualgen. Die Nahrung ist unmittelbar nach dieser rapiden Phagocytose in einer einzigen, sehr großen Vakuole eingeschlossen, die fast den ganzen Ciliaten ausfüllt. Im Verlaufe der folgenden Stunde vesikuliert diese große Nahrungsvakuole über Zwischenstufen zu einer Vielzahl von Vakuolen mit 1–2 m Durchmesser. Gleichzeitig erfolgt eine Kondensierung des Vakuoleninhaltes. Erst zu diesem Zeitpunkt setzt die Verdauung der Nahrung ein, wie an Hand von zahlreichen Dictyosomen belegt wird, die nun in unmittelbarer Nähe der Nahrungsvakuolen nachzuweisen sind. Durch die Vesikulation der großen Nahrungsvakuole in kleinere Einheiten sowie durch die Kondensierung der Nahrung wird bewirkt, daß die über Lysosomen in die Nahrungsvakuolen abgegebenen Verdauungsenzyme optimal eingesetzt werden. Nach Beendigung der Verdauung liegen viele leere Vakuolen vor, die durch eine stark gefaltete Kontur gekennzeichnet sind. Diese Vakuolen gehen allem Anschein nach wieder in den Membranhaushalt der Zelle ein.

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On the digestion in Pseudomicrothorax dubius Mermod (Ciliophora) vesiculation of the food vacuole following phagocytosis

Summary The gulper Pseudomicrothorax dubius ingests a large volume of filamentous blue-green algae within 1–2 min. Immediately after this rapid phagocytosis, the food is enclosed in a single, extremely large food vacuole, which fills up the ciliate almost entirely. During the following hour this giant food vacuole vesiculates. Finally numerous small vacuoles are present, 1–2 m in diam. Simultaneously the content of the vacuoles is noticeably condensed. At this time the digestion of the food starts as is indicated by numerous dictyosomes, which now surround the periphery of the food vacuoles. Due to both, the prior vesiculation of the food vacuole and the condensation of the food, the digestive enzymes can act very effectively. After 6–8 hours, when the digestion of the food is finished, numerous empty vacuoles are found. Each is characterized by a highly irregular, convoluted outline. Apparently these vacuoles are eventually recycled to the membrane pool of the cell.

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Für umsichtige und sorgfältige technische Assistenz danke ich Frl. A. Rüskens. Die Deutsche Forschungsgemeinschaft unterstützte diese Untersuchung durch die Sachbeihilfe Ha 818/7     

Involvement of smooth and coated vesicles in the function of the contractile vacuole complex of some cryptophycean flagellates

The contractile vacuole complex of cryptophycean flagellates comprises the contractile vacuole, a pore and a vesicular spongiome. A minority of spongiome vesicles bear a 15-nm coat on the cytoplasmic surface of the membrane. The coat superficially resembles a clathrin coat. The majority of vesicles are smooth surfaced. Both types of vesicles are found at the same time. Smooth vesicles can be seen in profile suggesting vesicle-vesicle and vesicle-vacuole fusion. It is suggested that smooth vesicles are involved in the segregation of fluid from the cytoplasm and in filling the vacuole. Coated elements exist only as independent vesicles and as coated pits in the contractile vacuole membrane. There is no evidence of fusion of coated vesicles. It is suggested that coated vesicles function to retrieve specific membrane components from the contractile vacuole.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Radioimmunologic proinsulin determination in the serum using an "insulin-catabolizing protease" from the rat liver]

Proinsulin determinations in human serum are of particular interest, because proinsulin represents only a fraction of the biological activity of insulin. Proinsulin like components are determined by means of an "Insulin Degrading Protease" (ISP) which degrades insulin into non-radioimmunoassayable fission products. The radioimmunoassay before and after incubation with this enzyme provides values for the total-insulin and the proinsulin. The preparation of the ISP is done by homogenization and ultracentrifugation of fresh liver tissue followed by dialysation and dryfreezing. After further concentration by adsorption to a calcium-phosphate-gel the ISP degrades pure porcine insulin within 20' down to a rest of 9%, but only 7% of pure porcine proinsulin is altered. The proinsulin values provided this way are reproducible and exact enough for the clinical use. They correspond largely to those methods using chromatographic columns. In 13 persons the proinsulin fraction of the total insulin after stimulation with glucose and tolbutamid has been registered. The proinsulin shows in the oral glucose tolerance test compared to insulin a lower and delayed increase. After tolbutamid only minor changes of proinsulin values have been seen. As long as it is difficult to prepare a proinsulin specific antibody for a direct proinsulin radioimmunoassay, the ISP-method is even qualified for extensive proinsulin determinations in human serum.