Ammonium-limited growth and uptake by Oscillatoria agardhii in chemostat cultures

Growth of Oscillatoria agardhil was studied in ammonium-limited chemostat cultures, at various dilution rates (=growth rates, μ). The uptake kinetics for ammonium of nitrogen (ammonium or nitrate)-limited chemostat cultures also was investigated. The kinetics of ammonium-limited growth could be adequately described by both the Monod and Droop equations, and were closely similar to the nitrate-limited growth kinetics of this species. The uptake kinetics for ammonium showed similarities as well as differences with the uptake kinetics for nitrate. The similarities were apparent in the uptake capacity values for ammonium and nitrate , which were identical, high and independent of μ. The differences were to be found in the half-saturation constants for ammonium uptake and nitrate uptake , the former being hardly influenced by μ. A consitutive, high affinity, system is likely to operate in the uptake and assimilation of ammonium by nitrogen-limited O. agardhii. The use of ammonium uptake parameters in studies of growth-limiting factors in nature can provide information as to whether a nitrogen-limitation prevails in natural habitats of this species.     

Fine structure of the ‘Wulst’ region of the domestic fowl in organotypic cultures

Summary Parts of the Wulst region of the chick embryo brain were maintained for 39 days in vitro. Processes of adjacent glial cell form zonulae occludentes and desmosomal junctions in the uppermost stratum of the cultures. Subjacent to this layer, in the neuropil, axodendritic synapses are abundant. 10–20 m below the surface the perikarya of glial cells and neurons are found. The latter form small clusters, plasma membranes of contiguous cells being directly apposed to each other. Axosomatic synapses terminate on the perikarya. Occasionally one terminal synapses on two nerve cells simultaneously. Two types of cilia arise from basal bodies in the cytoplasm of nerve cells. Type I is a slender protrusion of about 0.5 m in diameter, extending into the neuropil. On transverse sections it displays a 9 + 0 pattern of organization of paired micro tubules proximally, and an 8 + 1 configuration more distally. The length of the cilium is approximately 7 m. Type II cilia also originate in the neuronal cytoplasm. The structure of the proximal portion is identical with that of type I cilia. Toward the tip, however, the type II cilium is characterized by a bulbous enlargement which is filled with loosely folded membranes. The fine structural details of this cilium correlate closely to the outer segments of retinal photoreceptor cells during differentiation. The possible role of a light receptor in the Wulst region of birds, controlling biological rhythms, is discussed. Acknowledgements. The authors wish to thank Mrs. H. Frenk for her expert assistance in tissue culture and electron microscopic techniques     

Stereological and biochemical analysis of prolactin and growth hormone secreting rat pituitary cells in culture

Summary The secretion of prolactin is increased by treatment of prolactin producing rat pituitary cells with the hypothalamic tripeptide thyroliberin. To investigate the underlying mechanisms we used three closely related rat pituitary tumor cell strains (GH₁2C₁, GH₃ and GH₄C₁), which synthesize and spontaneously secrete prolactin and/or growth hormone. Growth hormone and prolactin released into the culture medium over a period of 24 h were measured by radioimmunoassay. Initial rates of synthesis were measured by immunoprecipitation of intracellular growth hormone and prolactin after incubation of cell cultures with ³H-leucine. The observed increase in prolactin synthesis and release was correlated with morphological effects of thyroliberin treatment. The volume density of Golgi complexes and the volume and surface densities of rough endoplasmic reticulum were compared in untreated cells and thyroliberin treated cells. As normal distribution could not be assumed, the non-parametric rank test of Wilcoxon was used whereby the densities calculated for each cell section were ranked. Alle three morphological parameters increased after thyroliberin treatment in cells secreting prolactin only (GH₄C₁), implying that the increase of prolactin secretion, at lest in part, is due to increased prolactin synthesis.     

Specific synaptic systems in reaggregated spherules from dissociated chick cerebellum cultivated in vitro

Summary Studies were performed on spherules of approximately 100–300 m in diameter obtained from in vitro cultures of reaggregates of embryonic fragments of cerebellum from 10–12 day-incubated chick embryos, dissociated with trypsin and cultivated in a rotating shaker for a maximum of 21 days. The differentiated neurons within these spherules included a few Purkinje cells, many granule cells and type II Golgi cells, as well as many glial cells. Zones rich in synaptic knobs and other simple synaptic structures as well as complex synaptic systems with numerous active points of contact, were visible in various parts of the spherule. Typical glomeruli consisting of a varicosity or rosette joined to the dendritic claws of the granule cells, and en marron systems with perikarya of type II Golgi cells were easily recognised. The complete absence of extracerebellar afferents confirms that both the granule and Golgi cells are capable of making synaptic connections with afferents different from those normally formed by extracerebellar mossy or climbing fibres.The experimental findings confirm that the recipient neurons determine the specific synaptic pattern regardless of the nature of the afferents, and furthermore demonstrate that the clinging activity of the recipient neuron determines the synaptogenic behaviour of nervous pathways.This work was supported by a grant from the Consiglio Nazionale delle Ricerche. Technical assistance was given by D. Scorsini for the ultrathin sectioning and electron microscopy, and by G. Gentili for the in vitro preparation and micrographs     

Metabolism of phenylalanine and tyrosine in tobacco cell lines resistant and sensitive to p-fluorophenylalanine

Chromatography of soluble polyphenols of p-fluorophenylalanine-sensitive and -resistant tobacco cells revealed that the 10-fold increased level found in the resistant line was largely due to the accumulation of two unidentified polyphenols. The uptake of Phe-[U-¹⁴C] and Tyr-[U-¹⁴C] by the resistant line was ca 10 % that by the sensitive line. About 90 % of the phenylalanine-[¹⁴C] which was taken up by both cell lines could be accounted for as free phenylalanine in protein, soluble polyphenols or CO₂. The fate of Tyr-[¹⁴C] was similar to that of phenylalanine except that the incorporation was into insoluble polymeric forms of polyphenols rather than into soluble polyphenols. The resistant line incorporated 9-times more phenylalanine-[¹⁴C] into soluble polyphenols than did the sensitive line. The different ¹⁴C-aromatic amino acid accumulation and incorporation patterns noted with the two cell lines indicates that there are different active pools. Differential uptake rates by the two cell lines might affect the distribution of the absorbed amino acid among the different pools.     

Glycine cleavage and generation of one-carbon units in Euglena gracilis

Euglena gracilis Klebs (strain Z) was maintained in division synchronized autotrophic culture, receiving either air (low CO₂) or 5% CO₂ in     

The differential effects of TCA-cycle acids on the growth of plant cells cultured in liquid media containing various nitrogen sources

Alfalfa (Medicago sativa L. cv. Canadian No. 1), tobacco (Nicotiana tabacum L. var. humilis) and wheat (triticum monococcum L.) cells were grown in a defined, liquid medium containing either ammonium sulfate, L-glutamine or potassium nitrate as the sole nitrogen source, and the effects of the tricarboxylic-acid (TCA) intermediates, citrate and -ketoglutarate (5, 10, 15 mM), on the growth (dry-weight increase) of these cells was observed. The three cell suspension cultures exhibited a different growth response to the TCA-cycle intermediate supplied, depending upon the concentration of the additive and the nitrogen source. Citrate (5 mM) greatly enhanced growth of alfalfa and wheat cells in an ammonium-based medium but was less effective at higher concentrations, and in the case of alfalfa cells markedly inhibited growth. Tobacco cell growth was inhibited by all citrate concentrations tested. In contrast, all concentrations of -ketoglutarate used stimulated the growth of all three cell cultures in an ammonium-based medium. Alfalfa and wheat cells grown in an L-glutamine-based medium were influenced by citrate in a manner similar to that in ammonium-based medium. The growth of tobacco cells was slightly enhanced by 5 mM citrate but inhibited by higher concentrations. -Ketoglutarate, at all concentrations tested, was stimulatory to the growth of the cells of all three species in a glutamine-based medium, except for alfalfa cells which were inhibited at 15 mM. Both TCA-cycle acids inhibited the growth of alfalfa and tobacco cells grown on a nitrate-based medium whereas the growth of wheat cells was almost unaffected.     

Metabolism of benzo[a]pyrene in cell suspension cultures of parsley (Petroselinum hortense,Hoffm.) and soybean (Glycine max L.)

Cell suspension cultures of parsley and soybean were incubated for 38 h with ¹⁴C-labeled benzo[a]pyrene; autoclaved cultures were used as controls. Metabolites were isolated by a sequential extraction procedure and further studied by chromatography or by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The soluble metabolites amounted to 1–2.2% in the case of parsley cells, and 19–28% in the case of soybean cells. These metabolites varied in polarity, some being soluble in organic solvent or aqueous buffer while other metabolite fractions were soluble only in hot aqueous sodium dodecylsulphate. In addition, a significant amount of an insoluble metabolite fraction was isolated from the culture fluid as well as the cellular material of soybean suspension cultures.Abbreviations BP benzo[a]pyrene - SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Polyamines in Paul's Scarlet rose suspension cultures

Polyamine concentrations have been determined at intervals in suspension cultures of Paul's Scarlet rose cells during a culture period of 2 weeks. The mean concentrations of the putrescine, spermidine and spermine in the cells of the inocula were respectively 73, 70 and 13 nmol/g fresh weight. Putrescine at fitst increased with a peak (160 nmol/g) after 6 h, declined to a minimum (14 nmol/g) after 2–3 days, increased to a second peak (180 nmol/g) after 5–6 days, and then declined slowly to the concentration of the inoculum (taken on day 14). Spermidine rose slowly (×2.6) to a broad peak over 3–6 days (180 nmol/g), then declined slowly to the concentration in the inoculum. Spermine showed a rapid increase to a peak (130 nmol/g) after 2–3 days, and then declined rapidly, reaching the inoculum concentration by day 6. In one experiment the three amines showed a minor peak at day 11. Changes in spermine and RNA contents appeared to be correlated. DNA content reached a peak after that of the RNA (day 3) and did not appear to be correlated with the content of putrescine or the polyamines.