Electron-cytochemical localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31) in fungal cells

Summary New cytochemical method, based on biochemical experiments, was elaborated for the ultrastructural localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). The procedure was used to study the saprophytic submerged mycelium of the ascomycetous fungusClaviceps purpurea Tul. producing clavine alkaloids. The pelleted mycelium was fixed in ice cold 3% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 and washed repeatedly in the same cold buffer. The reaction mixture contained 100 mM Tris-HCl buffer pH 9.0, 10 mM phospho(enol)pyruvate, 30 mM sodium potassium tartrate, 3 mM Pb(NO₃)₂, 60 mM MgCl₂ and 30 mM NaHCO₃. Enzyme activity was localized in vacuoles, particularly inside lipid globules (spherosomes) and less frequently in membranous vesicles. Acetyl-CoA activated PEP-carboxylase both in cell free extracts and in the cytochemical staining. Aspartate inhibited the enzyme in the biochemical assay with coupled malate dehydrogenase system; the cytochemical reaction was not influenced, probably due to the interference of asparagine synthase (EC 6.3.1.1).     

The cytotoxic and cytocidal effect of colicin E3 on mammalian tissue cells

The plasma membrane of mammalian cells can mediate the cytotoxic and cytocidal effects of colicin E3. As little as 10² lethal units of purified colicin E3 per cell exert a pronounced cytocidal effect on human epithelial HeLa cells and as little as 10⁴ lethal units per cell also on line L mouse fibroblasts in tissue culture. Cells in complete monolayers are rapidly killed, become spherical and shrink, they are detached from the support and finally autolyzed. The percentage of killed cells in both lines is directly proportional to the multiplicity of colicin used. Theld ₅₀ for HeLa cells is about 30 times lower than for L cells. At the multiplicity of 10⁵ l.u., usually 100 % HeLa cells and 90 % L cells are killed in 2–3 days. Purified colicins E2 and D have no demonstrable cytological effect on HeLa cells, although DNA synthesis in L cells appears to be partly inhibited by colicin E2. The profound effect of colicin E3 on mammalian cells could be interpreted in a similar way as in bacteria,viz. as a specific cleavage of rRNA.     

The effect of ultrasound and its combination with radiation on the genetic material ofSaccharomyces cerevisiae

Ultrasonication at 20 kHz, intensity 35 W/cm² and amplitude 15–25 μm of a diploid strain ofSaccharomyces cerevisiae was found to act as a weak mutagen with maximum efficiency at the 20 % survival of the cells. Under these conditions, the frequency of reversion of the suppressible allele ilv1-92 increased ten times, the frequency of mitotic gene conversion four times. Doses leading to survivals lower than 20 % led to a slight increase in the frequency of cytoplasmic respiration-deficient mutants. Submutagenic doses applied immediately after γradiation or UV light did not substantially increase the effect of these physical agents on the genetic material of the yeast strain investigated. Application of ultrasound prior to UV radiation did not considerably influence the effect of the radiation either.     

The effect of calcium on the growth ofChlorella andScenedesmus

The paper deals with the problem of the significance of Ca²⁺ for the growth of algae, its participation in the process of nitrate and ammonium nitrogen utilization, and with the possibility of substituting Sr²⁺ for it. It was revealed that calcium represents a microbiogenous element for the algae tested, which takes part in the utilization of nitrate nitrogen. It cannot be replaced by strontium especially at a high growth rate of mixotrophically cultivated algae (ina glucose medium).     

Uptake of microperoxidase by segmenting rat ova in vitro

Summary Uptake and incidence of microperoxidase (Sigma) in the rat ova during cleavage at the 1-, 2-, 8-cell and blastocyst stages were studied after 30 min incubation with the enzyme (molecular weight of 1,900 and size of molecule of 2 nm).Evidence was furnished of the presence of a small amount of reaction product of microperoxidase in the zona pellucida and its local concentration in some parts of the perivitelline space. The larger part of surface of the ovum, however, was free of microperoxidase. In the cytoplasm microperoxidase was found in pinocytotic vesicles, less frequently in large vacuoles and starting with the eight-cell stage in secondary lysosomes. The largest amount of microperoxidase was ascertained at the stage of blastocyst, chiefly in the cells of the trophoblast. In the cells of the embryoblast, on the contrary, microperoxidase was found but occasionally. The reaction product of microperoxidase was also present in the intercellular space of ova in negligible amount, and likewise on the side of blastocysts' cavity. In comparison with the ingestion of horseradish peroxidase the incidence of microperoxidase in the segmenting rat ova was less frequent.     

Origin of sclerotia-like cells in submerged Claviceps purpurea producing clavine alkaloids

Ultrathin sectioning of submerged mycelium of Claviceps purpurea Tul. producing clavine alkaloids revealed yeast-like budding resulting in asexual sporesblastospores. These deciduous spores were born by extended hyphal cells and retained the same ultrastructure of cell organelles. Both the extended hyphae and the blastospores resembled the cells of ergot sclerotial tissue. A surface culture of C. purpurea Tul. producing no alkaloids was used as a reference.     

Kymatocalyx Herz. in Mittelamerika

Summary A second species of the hitherto monotypic genusKymatocalyx Herz. is described and figured in detail. The new combinationKymatocalyx dominicensis (Spruce) comb. nova is proposed.     

Glutamate decarboxylase activity in Trichoderma viride conidia and developing mycelia

Glutamic acid decarboxylase (GAD) activity was measured in homogenates of conidia and both submerged and aerial mycelia of Trichoderma viride. The GAD activity in conidia had a temperature optimum at 30 degrees C and a pH optimum at pH 4. GAD was stimulated by EDTA (2 mM) and was insensitive to treatment with calmodulin antagonists calmidazolium (10 microM) or phenothiazine neuroleptics (60 microM). Cyclosporin A (up to 300 microM) partially inhibited GAD in the homogenate, but not in the supernatant obtained after centrifuging the homogenate. Attempts to release GAD activity from the homogenate using high ionic strength, detergents, or urea failed. Freezing-thawing led to the partial increase of activity in the conidial homogenate. These results indicate that GAD is a membrane-bound enzyme. The highest specific activity of GAD was present in the mitochondrial/vacuolar organellar fraction. Germination of conidia in the submerged culture led to a temporary decrease in GAD activity. After prolonged cultivation, the activity displayed quasi-oscillatory changes. The stationary state was characterized by a high GAD activity. The presence of gamma-aminobutyric acid in the submerged mycelia was demonstrated. In surface culture in the dark, GAD activity increased in a monophasic manner until conidia formation. The illumination of dark-cultivated mycelia by a white-light pulse caused a dramatic increase in GAD activity. Light-induced changes were not observed in mutants with delayed onset of conidiation. In the dark or upon illumination by light pulse, the increase of GAD activity preceded the appearance of conidia. Thus, GAD activity in T. viride is closely associated with its developmental status and may represent a link between differentiation events and energy metabolism.