Replacement of potassium ions by ammonium ions in different micro-organisms grown in potassium-limited chemostat culture

The biomass concentration extant in potassiumlimited cultures of either Klebsiella pneumoniae or Bacillus stearothermophilus (when growing at a fixed temperature and dilution rate in a glucose/ammonium salts medium) increased progressively as the medium pH value was raised step-wise from 7.0 to 8.5. Because the macromolecular composition of the organisms did not vary significantly, this increase in biomass could not be attributed to an accumulation of storage-type polymers but appeared to reflect a pH-dependent decrease in the cells' minimum K⁺ requirement. Significantly, this effect of pH was not eviden with cultures in which no ammonium salts were present and in which either glutamate or nitrate was added as the sole nitrogen source; however, it was again manifest when various concentrations of NH₄Cl were added to the glutamate-containing medium. This suggested a functional replacement of K⁺ by NH ₄ ⁺ , a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 Å) and the ammonium ion (1.43 Å). At pH 8.0, and with a medium containing both glutamate (30 mM) and NH₄Cl (100 mM), cultures of B. stearothermophilus would grow without added potassium at a maximum rate of 0.7 h^-1. Under these conditions the cells contained maximally 0.1% (w/w) potassium (derived from contaminating amounts of this element in the medium constituents), a value which should be compared with one of 1.4% (w/w) for cells growing in a potassiumlimited medium containing initially 0.5 mM K⁺. Qualitatively similar findings were made with cultures of K. pneumoniae; and whereas one may not conclude that NH ₄ ⁺ can totally replace K⁺ in the growth of these bacteria, it can clearly do so very extensively.     

Qualitative and quantitative comparison of glucose transport activity and glucose transporter concentration in plasma membranes from basal and insulin-stimulated rat adipose cells.

Conditions are described which allow the isolation of rat adipose-cell plasma membranes retaining a large part of the stimulatory effect of insulin in intact cells. In these membranes, the magnitude of glucose-transport stimulation in response to insulin was compared with the concentration of transporters as measured with the cytochalasin-B-binding assay or by immunoblotting with an antiserum against the human erythrocyte glucose transporter. Further, the substrate- and temperature-dependencies of the basal and insulin-stimulated states were compared. Under carefully controlled homogenization conditions, insulin-treated adipose cells yielded plasma membranes with a glucose transport activity 10-15-fold higher than that in membranes from basal cells. Insulin increased the transport Vmax. (from 1,400 +/- 300 to 15,300 +/- 3,400 pmol/s per mg of protein; means +/- S.E.M.; assayed at 22 degrees C) without any significant change in Km (from 17.8 +/- 4.4 to 18.9 +/- 1.4 nM). Arrhenius plots of plasma-membrane transport exhibited a break at 21 degrees C, with a higher activation energy over the lower temperature range. The activation energy over the higher temperature range was significantly lower in membranes from basal than from insulin-stimulated cells [27.7 +/- 5.0 kJ/mol (6.6 +/- 1.2 kcal/mol) and 45.3 +/- 2.1 kJ/mol (10.8 +/- 0.5 kcal/mol) respectively], giving rise to a larger relative response to insulin when transport was assayed at 37 degrees C as compared with 22 degrees C. The stimulation of transport activity at 22 degrees C was fully accounted for by an increase in the concentration of transporters measured by cytochalasin B binding, if a 5% contamination of plasma membranes with low-density microsomes was assumed. However, this 10-fold stimulation of transport activity contrasted with an only 2-fold increase in transporter immunoreactivity in membranes from insulin-stimulated cells. These data suggest that, in addition to stimulating the translocation of glucose transporters to the plasma membrane, insulin appears to induce a structural or conformational change in the transporter, manifested in an altered activation energy for plasma-membrane transport and possibly in an altered immunoreactivity as assessed by Western blotting.     

Inhibition of insulin-stimulated glucose transport in rat adipocytes by nucleoside transport inhibitors

In isolated rat adipocytes, basal as well as insulin-stimulated 3-O-methylglucose transport was inhibited nearly completely (maximal inhibition: 95%) by the nucleoside transport inhibitors dipyridamole (IC50 = 5 microM), nitrobenzylthioguanosine (20 microM), nitrobenzylthioinosine (35 microM) and papaverine (130 microM). Transport kinetics in the presence of 10 microM dipyridamole revealed a significant increase in the transport Km value of 3-O-methylglucose (3.45 +/- 0.6 vs 2.36 +/- 0.29 mM in the controls) as well as a decrease in the Vmax value (4.84 +/- 0.95 vs 9.03 +/- 1.19 pmol/s per microliter lipid in the controls). Half-maximally inhibiting concentrations of dipyridamole were one order of magnitude higher than those inhibiting nucleoside (thymidine) uptake (0.48 microM). The inhibitory effect of dipyridamole (5 microM) reached its maximum within 30 s. The agent failed to affect insulin's half-maximally stimulating concentration (0.075 nM) indicating that it did not interfere with the mechanism by which insulin stimulates glucose transport. Further, dipyridamole fully suppressed the glucose-inhibitable cytochalasin B binding (IC50 = 1.65 +/- 0.05 microM). The data indicate that nucleoside transport inhibitors reduce glucose transport by a direct interaction with the transporter or a closely related protein. It is suggested that glucose and nucleoside transporters share structural, and possibly functional, features.     

Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

Summary Lymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.Animals were maintained under the guidelines laid down by the Faculty of Veterinary Medicine, State University, Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation) Abbreviations used: BCG, Bacillus Calmette-Guerin; BOSCC, bovine ocular squamous cell carcinoma PBL peripheral blood leukocytes; PPD, purified protein derivative of Mycobacteria; DTH, delayed type hypersensitivity Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen     

The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: Study by high-performance liquid chromatography

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F₁ ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima,K _m values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg²⁺. Although the equations giving the reaction rate are similar in the two cases, the calculated values ofK _m are widely different. The value obtained from the classical mechanism does not agree withK _D , the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na⁺ is more easily explained as a competition between this ion and the activating Mg²⁺, than by the classical mechanism.     

Simultaneous measurement of changes in red and blue fluorescence in illuminated isolated chloroplasts and leaf pieces: The contribution of NADPH to the blue fluorescence signal

A newly developed nitrogen laser fluorimeter insensitive to actinic illumination was used to follow simultaneously the light induced changes in red and blue fluorescence of intact isolated spinach chloroplasts and leaf pieces. The recorded variable blue fluorescence was linked to a water soluble component of intact isolated chloroplasts, depended on Photosystem I, and was related to changes in carbon metabolism. From the comparison of changes in intact and broken chloroplasts and from fluorescence spectra under different conditions, it was concluded that the variation in NADPH was the major cause for the changes in blue fluorescence. This study opens a path towards continuous and non-destructive monitoring of NADPH redox state in chloroplasts and leaves.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - DLGA DL-glyceraldehyde - FNR ferredoxin-NADP reductase - FWHM full width at half maximum - LED light emitting diodes - OAA oxaloacetate - q_N non-photochemical quenching - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - q_P photochemical quenching - PPFD photosynthetic photon flux density - Q_A primary quinone acceptor of Photosystem II Preliminary results of this work were presented at the First Conference on the Physiology and Biochemistry of high Mountain Plants, 2–3 July 1992, Villar d'Arene, France.     

Practical suggestions for successful immunoenzyme double-staining experiments

Summary Many methodologies exist to perform an immunoenzyme double staining. Hence, the practical problem arises as to which of these methods is optimal for one's own experimental design. A process of selection is described which is derived from our own practical experience. First, a general strategy is outlined for the handling of tissue sections to be used for multiple staining methods. Secondly, the selection of an appropriate immunoenzyme double-staining concept is made using a flow chart. Thereafter we give criteria for the definitive selection of an immunoenzyme double-staining protocol based on the characteristics of the tissue or cell type under study. Particular attention is given to the selection of appropriate detection systems, applying enzymes or gold particles, and good contrasting colour combinations. The problems of visualizing co-localization using immunoenzyme double staining are dealt with, and suggestions are made to adapt the method, if necessary, in order to optimize it.This paper (in modified form) is part of the thesis of C. M. van der Loos: Free University Press 1992, Amsterdam, The Netherlands (ISBN 90-5383-081-2).     

Therapy of bovine ocular squamous-cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up

We have tested the therapeutic potency of peritumorally injected low doses of interleukin-2(IL-2). Seventy tumours of the bovine ocular squamous-cell carcinoma (BOSCC), 1–3 cm in diameter, were treated with 5000, 20 000 or 200 000 U IL-2 from Eurocetus (Chiron) to find the optimal dose for treatment. Injections were given peritumorally on Monday to Friday on 2 consecutive weeks. The size of the tumours was measured before treatment and 1, 3, 4, 9 and 20 months after treatment. After 9 months complete regression was observed in 89% of the tumours treated with 5000 U IL-2, 80% treated with 20 000 U and 67% treated with 200 000 U. After 20 months, there was complete regression of 35%, 31% and 67% of the tumours respectively. The 9-and 20-month results of the 200 000-U treatment are significantly better than those of the 5000-U and 20 000-U treatments taken together. This protocol may be useful to treat advanced inoperable tumours (e.g. of the nasopharynx or skin) of human patients.