Enhancement of specific nitrogenase activity inAzospirillum brasilense andKlebsiella pneumoniae,inhibition inRhizobium japonicum under air by phenol

Specific nitrogenase activity inAzospirillum brasilense ATCC 29145 in surface cultures under air is enhanced from about 50 nmol C₂H₄·mg protein^-1·h^-1 to 400 nmol C₂H₄ by the addition of 1 mM phenol. 0.5 and 2 mM phenol added increase the rate 5-fold and 4-fold. This enhancement effect is observed only between 2 and 3 days after inoculation, with only a small reduction of the growth of the cells by the phenol added. In surface cultures under 1% O₂, nitrogenase activity is slightly reduced by the addition of 1–0.01 mM phenol. Utilization of succinate is enhanced during the period of maximum enhancement of nitrogenase activity by 60% by addition of 1 mM phenol. The cells did not produce¹⁴CO₂ from [U-¹⁴C] phenol, neither in surface cultures nor in liquid cultures and less than 0.1% of the phenol was incorporated into the cells. A smaller but significant enhancement of nitrogenase activity by about 100% in surface cultures under air was found withKlebsiella pneumoniae K 11 after addition of 1 mM phenol. However, inRhizobium japonicum 61-A-101 all phenol concentrations above 0.01 mM reduced nitrogenase activity. With 1 mM phenol added activity was reduced to less than 10% with no effect on the growth in the same cultivation system. With thisRhizobium japonicum strain significant quantities of phenol (25 mol in 24 h by 2·10¹² cells) were metabolized to¹⁴CO₂, with phenol as sole carbon source. WithAzospirillum brasilense in liquid culture under 1% and 2% O₂ in the gas phase, no enhancement of nitrogenase activity by phenol was noticed.     

Computer analysis of two-dimensional gels

New methods and computer programs are described which enable one to analyze autoradiograms produced by two-dimensional gel electrophoresis. These programs are completely automatic with respect to finding spots resolved by such gels and quantitating the radioactivity in them. Semiautomatic programs have also been developed to match the spot patterns of different autoradiograms, and to follow the synthesis of any individual polypeptide through a series of gels.     

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).     

Oxygen solubilities in fermentation fluids

Summary Fermentation media consist of a large number of chemicals whose composition undergoes alteration during the course of fermentation. As a result of this, conventional methods and correlations for oxygen solubility measurement and prediction do not apply in these systems. Using a physical method, oxygen solubilities were measured in simulated chemical systems and in fermentation broths. Sugars, salts, and fermentation products were identified as major factors influencing oxygen solubility. Salt effect was correlated with electrical conductivity of the medium, which was easy to measure during fermentation. For mixtures and for fermentation medium, individual influences were found to be log-additive in accordance with Danckwerts (1970).     

Die Pulvillen vonCalliphora erythrocephala (Diptera,Brachycera) als Adhäsionsorgane

Zusammenfassung Die Pulvillen vonCalliphora erythrocephala wurden licht und fluoreszenzmikroskopisch, raster- und transmissions-elektronenmikroskopisch untersucht. Das Adhäsionssekret wurde dünnschichtchromatographisch mit dem Körperoberflächen-Lipid verglichen.Die Pulvillen tragen auf der Ventralseite mehrere Tausend langer dünner Hafthaare mit sohlenartigen Endverbreiterungen. Die Kutikula des Pulvillus besteht hauptsächlich aus elastischen Kutikulatypen (Endo- und Mesokutikula) in verschiedenen, spezifischen Ausbildungsformen.Die Pulvillen enthalten ein sekretorisches Epithel ohne Ausführgänge, das Protein- und Lipideinschlüsse zeigt.Das Pulvillensekret ist eine schwerflüchtige, ölige Flüssigkeit, die nicht identisch ist mit dem Körperoberflächen-Lipid. Das Sekret wird in der homogenen Kutikala des Pulvillus gespeichert und über das Porenkanalsystem der Ventralseite nach außen geleitet.Der Zusammenhang zwischen der Struktur der Pulvillen und ihrer Rolle beim Adhäsionsvorgang wird diskutiert.

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The pulvilli ofCalliphora erythrocephala (Diptera, Brachycera) as adhesive organs

Summary The pulvilli ofCalliphora erythrocephala were examined by light microscopy, fluorescent light microscopy, SEM and TEM. The adhesive secretion was compared with the general surface lipid layer by means of thinlayerchromatography.On the ventral surface the pulvilli bare several thousand long, slender tenent hairs with solelike spatulate tips. The pulvillar cuticle is composed on the whole of elastic cuticle types (endocuticle, mesocuticle) with specific modifications of structure. The pulvilli contain a secretory epithelium without transport ducts which has lipid and proteinaceous inclusions.The pulvillar secretion is a slowly volatile oily liquid which is not identical to the general surface lipid. The secretion is stored in the homogeneous cuticle within the pulvillus and transported to the ventral surface via the pore canal system.The correlation between the structure of pulvilli and their function in the adhesion process is discussed here.

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Abkürzungen bl Basales Labyrinth - cv coated vesicle - db dense body - dlk Dichter Lamellenkörper - dw Dorsalwand - e Epithel - ef Endokutikulafasern in der Übergangszone (meso 2b) - endo 1 lamelläre Endokutikula - endo 2 homogene Endokutikula - endo 3 gelatinöse Endokutikula - ep Empodium - epi Epikutikula - exo Exokutikula - fr Fersenregion - fs Faltensaum - hh Hafthaare - hs Haarschaft - k Kutikula - kr Kralle - ku Kutikulinschicht - l Lumen - li Lipid - lr Dorsale Längsrippen - m Mitochondrium - mb Mesokutikulabalken in der Übergangszone (meso 2b) - meso 1 helle homogene Mesokutikula - meso 2a dunkle homogene Mesokutikula, kompakt - meso 2b Übergangszone, in der dunkle homogene Mesokutikulabalken mit faseriger Endokutikula verzahnt sind - mt Mikrotubulus - mvbd Dunkler multivesikulärer Körper - n Kern - pc Porenkanal - ps Proximalsklerit - pt Prätarsus - rer Rauhes endoplasmatisches Retikulum - s Schulter - sd Sehnendrüse - se Sehne - so Sohle - sr Saumregion - t Tubulus - t₅ Tarsalglied 5 - ta Tasche - td Tarsaldrüse - te Tarsalepithel - tg Taschengrund - tk Terminalkanäle - utr Unguitractor - v Heller Vesikel - va Proteinvakuole - w Wachsschicht - z Zementschicht     

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     

Inhibitory effects of mersalyl and of antibodies directed against smooth-muscle myosin on a calcium adenosine triphosphatase of the plasma membrane from mouse liver cells.

The effect of mersalyl and of antibodies, directed against smooth-muscle myosin and skeletal muscle myosin, on the (Ca²⁺ + Mg²⁺)-activated adenosine triphosphatase (Ca,Mg)ATPase) system of mouse liver plasma membranes has been studied. Antismooth-muscle myosin inhibited by 38.6% at optimum substrate concentration the (Ca,Mg)ATPase with a K_m of 0.88 × 10^?3m. Mersalyl (0.5 mm) also inhibited this enzyme, the percentage inhibition being 44.6% at optimal substrate concentration. These results suggest the presence of a smooth-muscle myosin-like protein in the plasma membrane of mouse liver cells which has an associated (Ca,Mg)ATPase activity.     

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.