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).     

Pyrus spinosa und ihre Hybriden in Südwestbulgarien

Pyrus spinosa Forsk. (=P. amygdaliformis Will.) kreuzt sich in beträchtlichem Masse mitP. pyraster Burgsd. und mitP. communis L. Es wurden drei Hybriden beschrieben, die zwar weniger, aber gut keimende Samen haben. Potentiell sind in der bulgarischen Population vonP. spinosa Merkmale aller infraspezifischen Taxa vertreten, doch nur zwei von ihnen kommen in grösserem Masse vor, das Vorkommen der weiteren drei ist nur gering. Merkmale des übrigen infraspezifischen Taxon kommen nur in Kombination mit Merkmalen anderer Taxa bei Übergangsformen vor. Vorläufig ist auch die Verbreitung vonP. spinosa und ihrer Hybriden in Bulgarien angegeben.     

The capability of the algaeChlorella vulgaris andScenedesmus obliquus of utilizing amino acids as the sole nitrogen source

The capability of utilizing 20 amino acids and 2 amides as the sole nitrogen source for growth was studied in two green algae (Chlorophyceae). A comparison was made of the growth rate of algae in a mineral nutrient solution containing nitrate as the nitrogen source, with that in the same solution in which nitrogen in the form of nitrate was substituted by an equivalent nitrogen amount in the form of various amino acids. In addition to this, another series of experiments was carried out in whioh both culture media were supplied with glucose. The results show that both algae utilize a series of amino acids in dependence of their structure (mostly 3-carbon amino acids). The growth rate ofChlorella in the presence of these sources is the same as in nitrate, that ofScenedesmus even much higher. In the cultures containing glucose both algal species exhibit a higher growth rate in the media with the nitrate nitrogen source than in those with amino acids (with the exception of glycine inScenedesmus).     

Influence of antilymphocyte and antipolymorphonuclear sera on the pyrogenic effect of scarlet fever toxin

The role played by lymphocytes in the pyretic response to scarlet fever toxin (ET) was studied in vivo using antilymphocyte serum (ALS). Two i.v. injections of ALS inhibited the pyretic response to a subsequent ET injection in rabbits. The course of endotoxin fever remained uninfluenced by ALS. Antipolymorphonuclear serum had no effect on the pyretic response to either of the toxins. Pretreatment with ALS also inhibited the skin reaction after i.d. injection of ET. These findings are further evidence a mediating role of lymphocytes in the biological effects of ET, among other things in the release of endogenous pyrogen.     

Microorganisms in the rhizosphere of wheat colonized by the fungusGaeumannomyces graminis var.tritici

The population of microorganisms in wheat rhizosphere changed in the presence of the fungus Gaeumannomyces graminis var. tritici causing the take-all of wheat. In the majority of cases when the soil was artificially contaminated by the fungus, both the number of bacteria in the rhizosphere and the bacteria/fungi ratio temporarily increased. At the beginning bacteria growing in the presence of NH4+ predominated, later bacteria utilizing organic N-substances prevailed. Pseudomonas fluorescens and the related species colonized the rhizosphere and the soil to a greater extent in the presence of G. graminis. The wheat rhizosphere with G. graminis was found to contain a higher level of the slime-producing bacterium Agrobacterium spp.; this microorganism occurred on hyphal surfaces (in hyphosphere) of both G. graminis growing in soil and Mucor spp. Changes in microbial populations in the wheat rhizosphere during the first stage of colonization by G. graminis can be partly explained by a simultaneous rhizosphere colonization by microorganisms which accompany this fungus in soil. In the period of increase in the number of bacteria in rhizosphere a temporary stimulation of wheat growth was observed.     

Evaluation of three E-rosette assays in melanoma patients

Summary The percentage of E-rosette-forming cells (E-RFC) was determined repeatedly in the peripheral blood of 43 patients with malignant melanoma. Three methods of E-rosette assaying were used: total E-rosette test, active E-rosette test, and 29° C E-rosette assay.Depressed E-RFC values were found in fewer assays than normal values. The mean values of E-RFC and the proportion of depressed E-RFC in patients in stage I did not differ from the values in healthy control persons whatever method of assay was used.The frequency of depressed E-RFC values was higher in advanced stages. Significant differences were demonstrated between stage I and stages II and III combined in the values for total and for active E-rosettes (P<0.01).The active E-rosette test and the 29° C E-rosette assay gave no more positive results (i.e., depressed E-RFC values) than the total E-rosette test in melanoma patients.     

Electron-microscopic localization of acetyl coenzyme A carboxylase in cells of Claviceps purpurea

Summary The ultrastructural localization of acetyl-CoA carboxylase activity was studied in two strains of the ascomycetous fungus Claviceps purpurea differing in the ergot alkaloid synthesis. Mycelia were harvested by centrifugation of saprophytic submerged cultures, fixed in cold 3% glutaraldehyde in 0.05 M cacodylate buffer pH 7.2 and washed repeatedly in the same buffer. The incubation medium of Yates et al. (1969) had to be modified in the molarity of ATP. The best results were obtained with a medium of the following composition: 50 mM cacodylate buffer pH 7.2, 4 mM ATP, 3.5 mM lead nitrate, 13.5 mM sodium citrate, 3.75 mM sodium bicarbonate, 1.25 mM manganese chloride, 0.4 mM acetyl-CoA and 2 mM biotin. The fixation is a prerequisite for a distinct localization. The enzyme activity was detected only in cells producing high amount of clavine alkaloids. It was confined to the membranes of endoplasmic reticulum and their derivatives: tonoplast of vacuoles, tiny vesicles and amorphous material inside vacuoles. The reaction product was very fine and localized in both leaflets of the membranes. The specificity of the reaction was confirmed by negative results in control preparations: boiled cells incubated in the complete medium, cells incubated in the medium supplemented with avidin or in the media from which either ATP, or acetyl CoA, or sodium bicarbonate, or biotin were omitted. It is suggested that the activity of acetyl-CoA carboxylase is linked to the synthesis of clavine alkaloid precursors which occurs in the endoplasmic reticulum and its derivatives.With technical assistance of J. Martínková