Inhibition of oxidative phosphorylation by Ca or Sr: A competition with Mg for the formation of adenine nucleotide complexes

Intramitochondrial Sr²⁺, similar to Ca²⁺, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca²⁺ and Sr²⁺ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca²⁺ or 5.0 mM Sr²⁺ when the concentration of Mg²⁺ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg²⁺ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca²⁺ or Sr²⁺ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg²⁺ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr²⁺ it can be concluded that intramitochondrial Ca²⁺ or Sr²⁺ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

Cellular retinoic acid-binding protein and its relationship to the biological activity of four synthetic retinoids in hamster tracheal organ culture

Summary The mechanism of action of retinoid in reversing keratinization in hamster trachea is yet unknown. The purpose of this study was to determine if cellular retinoic acid binding protein (CRABP) is present in tracheal epithelium following incubation in serum-free, vitamin A-deficient culture medium for 10 days, and if the effectiveness of a retinoid in reversing keratinization in organ culture is correlated with its ability to compete for CRABP sites. The cytosol prepared from tracheal cultures contained CRABP at a concentration of 2.61 pmoles per mg protein. Of the four retinoids with carboxyl end group selected for the study, two of the biological active retinoids competed for the CRABP sites. However, no correlation was observed between the biological activity of the inactive retinoids and their ability to associate with the CRABP sites. These results indicate that even though the action of retinoid may be mediated by retinoid binding protein, it cannot be used as a sole predicator of retinoid response in hamster trachea. This investigation was supported by Contract N01-CP-31012 and U. S. P. H. Grants CA30512 and CA32428, which were awarded by the Division of Cancer Etiology, National Cancer Institute, DHHS. Editor's Statement Tracheal organ cultures provide a useful model for the study of epithelial differentiation and carcinogenesis. Much attention has been given to the action of retinoids in this process. Mehta et al. demonstrate a lack of correlation between biological activity and specific cytosolic binding of members of this class of compounds, pointing out the need for a more complete biochemical understanding of the mechanism of action and active forms of retinoids in this and other systems in vivo and in vitro. David W. Barnes     

Channel formation in phospholipid bilayer membranes by the toxin ofHeminthosporium maydis,race T

Summary Southern Corn Leaf Blight is caused by a toxin produced by a virulent form ofHelminthosporium maydis (Race T). The toxin has been shown to uncouple oxidative phosphorylation and dissipate Ca²⁺ gradients in mitochondria isolated from susceptible, but not resistant, corn. The possibility that the toxin acted by increasing the permeability of membranes to ions was tested using a planar bilayer membrane system. Addition of the toxin to the bilayer system, under voltage-clamp conditions, resulted in stepwise increases in current across the phospholipid bilayer, a response characteristic for channel formers. Single-channel conductance in 1m KCl is 27 pS which corresponds to 1.7×10⁷ ions sec^–1 channel^–1 at 100 mV applied potential. The toxin channels are: (i) fairly uniform in conductance, (ii) ideally selective for K⁺ over Cl^–, and (iii) most conductive to H⁺. The channel showed the following selectivity for alkali metal cations: Rb⁺>K⁺>Cs⁺>Na⁺>Li⁺ (169731) based on the most frequently observed conductance in 1m chloride salts. The toxin showed no voltage dependence over the range of –100 to +100 mV. Channel formation was also a property of a synthetic analog (Cmpd IV) of the toxin. The ability of the native toxin to form channels may be a mode of toxin action on mitochondrial membranes from susceptible corn.     

Immunofluorescent and calcofluor white staining of developing tracheary elements inZinnia elegans L. Suspension cultures

Summary Xylogenesis has been studied in primary suspension cultures ofZinnia elegans L.: The wall patterns produced in culture closely resemble those described for intact tissues (annular, spiral, reticulate, scalariform, pitted). Using fluorescence microscopy and immuno-cytochemical techniques we have followed both the changes in wall deposition and microtubule organization during xylogenesis. Calcofluor white has been used to detect secondary wall deposition before it can be observed using either phase contrast or polarization optics. The development of tracheary elements can be divided into three stages: 1. microtubules grouped into bands without secondary wall deposition evident; 2. groups of microtubules subtending wall material only visible using Calcofluor white; 3. a complex microtubule pattern reflected by well developed wall thickenings detected using Calcofluor, phase contrast and polarization optics.     

Stimulation of intestinal adenylate cyclase by cholera toxin in malnourished rats

The stimulation of intestinal adenylate cyclase by cholera toxin (CT) was studied in normal and malnourished rats 4 to 24 hr after a 30-min incubation of intestinal loops with the toxin. Whereas in control rats the enzyme activity returned to basal levels after 12 hr of incubation, in malnourished rats the activity of the enzyme remained significantly elevated even after 24 hr of the initial incubation. Malnourished animals had a reduced turnover rate of intestinal cells as determined by thymidine kinase activity. The delayed turnover of intoxicated cells may account for continuous activation of mucosal adenylate cyclase and possibly for prolongation of diarrhea in malnutrition.     

Kinetics of Micronucleus Expression In Synchronized Irradiated Chinese Hamster Ovary Cells

The kinetics of expression of radiation-induced micronuclei (MN) in synchronized Chinese hamster cells (CHO) was examined. the purpose of the study was to determine if the cell cycle distribution of a population significantly influences the levels of radiation induced MN, thereby obscuring the exact quantification of the radiation effect. Cells were synchronized by centrifugal elutriation, irradiated, and then different phases of the cell cycle were examined for: cell cycle progression, division probability, and temporal expression of MN. the results demonstrate that the time interval for maximal MN expression is long enough that the position of cells in the cell cycle and radiation induced division delays do not prevent the majority of cells from completing their first post-irradiation mitosis, therefore, expressing MN. By following the progression of synchronized cell populations by flow cytometry and also examining the time of division of individual cells for 24 hr after irradiation, we observed that the maximum number of cells from all phases of the cell cycle are in their first post-irradiation interphase at that time, thus explaining the MN results.     

Wheat germ agglutinin-resistant variant of EL4 containing altered oligosaccharides as a target cell for cytotoxic T cells

A lectin-resistant variant of the murine EL4 lymphocytic leukemia cell line was selected in the presence of wheat germ agglutinin for low levels of cell-surface sialic acid. H-2Kb was the major internally radiolabeled H-2b molecule on the cell-surface of WD1, and it was not sialylated, as determined by two-dimensional gel analysis. Endo-beta-N-acetylglucosaminidase H treatment of the WD1 membrane fractions suggested that the oligosaccharides on the cell-surface H-2Kb molecule were complex, but nonsialylated. Monoclonal antibody inhibition of the allogeneically primed cell-mediated cytotoxicity (CMC) reaction indicated that the T cells (BALB/c anti-EL4; H-2d anti-H-2b) were specific only for the H-2Kb target cell antigen. These WD1 variant cells were used as targets in the CMC assay using anti-H-2Kb T cells and compared with the parent EL4 in vitro line. The change in the cell-surface oligosaccharide did not affect the susceptibility to lysis by the cytotoxic T lymphocytes even though there were 2.5-fold more H-2Kb antigens on the WD1 variant cell (1.5 X 10(5) sites/cell) than on the parent EL4 in vitro cell (5.9 X 10(4) sites/cell). It was possible to isolate highly purified preparations of H-2Kb from either the EL4 or the WD1 line using a monoclonal antibody affinity column. Interestingly, the variant WD1 cell would no longer grow in the peritoneal cavity of the syngeneic C57BL/6 mouse.     

Production,by filamentous,nitrogen-fixing cyanobacteria,of a bacteriocin and of other antibiotics that kill related strains

Colonies of sixty-five filamentous cyanobacteria were screened for the production of temperate phages and/or antibiotics on solid medium. None of them was observed to release phages. However, seven N₂-fixing strains were found to produce antibiotics very active against other cyanobacteria. The antibiotic produced by Nostoc sp. 78-11 A-E represents a bacteriocin of low molecular weight. Nostoc sp. ATCC 29132 appears to secrete, together with an antibiotic, a protein that inhibits its action.