Plastid KEA-type cation/H+ antiporters are required for vacuolar protein trafficking in Arabidopsis

Arabidopsis plastid antiporters KEA1 and KEA2 are critical for plastid development, photosynthetic efficiency, and plant development. Here, we show that KEA1 and KEA2 are involved in vacuolar protein trafficking. Genetic analyses found that the kea1 kea2 mutants had short siliques, small seeds, and short seedlings. Molecular and biochemical assays showed that seed storage proteins were missorted out of the cell and the precursor proteins were accumulated in kea1 kea2. Protein storage vacuoles (PSVs) were smaller in kea1 kea2. Further analyses showed that endosomal trafficking in kea1 kea2 was compromised. Vacuolar sorting receptor 1 (VSR1) subcellular localizations, VSR–cargo interactions, and p24 distribution on the endoplasmic reticulum (ER) and Golgi apparatus were affected in kea1 kea2. Moreover, plastid stromule growth was reduced and plastid association with the endomembrane compartments was disrupted in kea1 kea2. Stromule growth was regulated by the cellular pH and K⁺ homeostasis maintained by KEA1 and KEA2. The organellar pH along the trafficking pathway was altered in kea1 kea2. Overall, KEA1 and KEA2 regulate vacuolar trafficking by controlling the function of plastid stromules via adjusting pH and K⁺ homeostasis.     

Equilibrium study on the binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI)

The binding between thermolysin and its specific inhibitor, talopeptin (MKI), was found to show a fluorescence increase when excited at 280 nm and 295 nm, and a difference spectrum characterized by two peaks at 294 nm and 285 nm with a shoulder around 278 nm, indicating a microenvironmental change in tryptophan residue(s) of thermolysin and/or talopeptin. The inhibitor constant of talopeptin against thermolysin, Ki, was determined over the pH range 5-9 from the inhibition of the enzyme activity towards 3-(2-furylacryloyl)-glycyl-L-leucine amide (FAGLA) as a substrate. The dissociation constant of thermolysin-talopeptin complex, Kd, determined directly from fluorometric titration was in good agreement with the inhibitor constant, Ki, between pH 6 and 8.5. The pH dependence of Ki and Kd suggested that at least two ionizable groups of thermolysin in their protonated forms are essential for the binding between thermolysin and talopeptin. The temperature dependence of K1 at pH 5.5 indicated that the binding is largely exothermic (delta H degree = -12 kcal/mol) and essentially enthalpy-driven.     

Effect of dibutyryl cyclic AMP on collagen synthesis in a clonal osteoblast-like cell line derived from newborn mouse calvaria

The effects of dibutyryl cyclic AMP (DBcAMP) and related compounds on collagen synthesis in a clonal osteoblast-like cell line, MC3T3-E1, were investigated. The addition of DBcAMP to cultures increased the hydroxyproline content of the cells. It also enhanced the incorporation of labeled proline into collagen and elevated the activity of prolyl hydroxylase, an enzyme involved in collagen synthesis. These effects were observed at concentrations of 0.1 to 2 mM DBcAMP. 8-Bromo cyclic AMP also increased the hydroxyproline content of the cells, while sodium butyrate and dibutyryl cyclic GMP had no such effect. These results suggest that the intracellular accumulation of cyclic AMP in osteoblasts leads to their active production of collagen, a major component of the organic matrix of bone.     

Properties of androphilic proteins in cytosols of human benign prostatic hypertrophy.

Androphilic proteins in the cytosol from the human benign prostatic hypertrophy are separated into two fractions by Sephadex chromatography; void volume fraction and IgG fraction which was eluted near the site of hIgG. In the present study, properties of these two androphilic proteins were compared. Association constants of these proteins were in the order of 10(9) M-1. However, the binding capacity of the former was smaller than that of the latter. These two androphilic proteins well bound to nuclei, and the high-affinity and saturable binding to nuclei was observed in the 3H-dihydrotestosterone-IgG fraction complex, while binding of 3H-dihydrotestosterone-void volume fraction complex to nuclei was low affinity and unsaturable. The binding of the complexes to chromatin seems to be of low affinity and nonsaturable. These androphilic proteins did not bind to calf thymus DNA. Salt extractability of the bound void volume fraction after incubation with nuclei was not different from that of the bound IgG fraction. It was observed that the chromatographic behavior of the androphilic protein in IgG fraction was changed after incubation with nuclei.     

Interaction of amylose and other α-glucans with hydrophobic fluorescent probe (2-p-toluidinylnaphthalene-6-sulfonate)

Fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was enhanced in the presence of maltooligosaccharides, amylose, and other α-glucans. The dependence of relative TNS fluorescence intensity per glucose unit on chain length of oligosaccharides was examined. The values of binding constant and thermodynamic parameters, assuming the 1:1 complex for TNS-amylose (number-average degree of polymerization, DP_N = 17), were determined by the fluorescence titration. The values of thermodynamic parameters for 1:1 complex formation of TNS-α- and β-cyclodextrins were also determined and compared with those of TNS-amylose (DP_N = 17). The fluorescence intensity of TNS in the presence of amylose (DP_N = 600) decreased by the action of glucoamylase and taka-amylase A. The fluorescence of TNS-amylose (DP_N = 17) system increased with the increased ionic strength. In the presence of pullulan, TNS fluorescence was also enhanced and decreased by the action of pullulanase. Amylopectin enhanced TNS fluorescence rather more strongly than amylose (DP_N = 17) at the same concentration. In the presence of dextran, the fluorescence of TNS was scarcely enhanced. The degree of fluorescence enhancement of TNS in the presence of α-glucans seems to reflect the structures of α-glucans in solution, since TNS fluorescence is enhanced in the hydrophobic environment or by the disturbance of free intramolecular rotation.     

Androphilic proteins in cytosols of human benign prostatic hypertrophy.

To examine the properties of androphilic proteins in human benign prostatic hypertrophy, the binding capacity and affinity of the proteins were determined after acetone-treatment, ammonium sulfate precipitation and chromatographies of DEAE and Sephadex G-200. Androphilic proteins in the extract of acetone-dried cytosol from the hypertrophic human prostate was precipitated at 30-50% saturation of ammonium sulfate. The binding of this fraction to dihydrotestosterone and testosterone was high affinity, but the binidng to estradiol-17 beta was the one of non-specific. Androphilic proteins in the 30-50% fraction were eluted from DEAE-cellulose column by buffer containing 0.05 M KCL. On Sephadex G-200 chromatography of 30-50% fraction, the androphilic proteins were observed in three peaks; one was eluted in the void volume and other two were eluted at the sites of IgG and albumin. The amount and ratio of proteins eluted in the void volume and the site of IgG from Sephadex G-200 column were variable in individual tissue samples. The chromatographic behavior of the 30-50% fraction in Sephadex G-200 was not changed significantly by introducing 0.4 M KCl in the system. Polyacrylamide gel electrophoresis was applied for further separation of the proteins.     

Karyological characterization of a human lymphoblastoid cell line resistant to 6-thioguanine

Summary A mutant human lymphoblastoid cell line, Raji-TG, resistant to 10g 6-thioguanine (TG)/ml was produced from wild-type cells after exposure to ethylmethane sulfonate. The Raji-TG cells showed their failure to incorporate ³H-hypoxanthine, only 2% as much hypoxanthine guanine phosphoribosyl transferase (HPRT) activity as wild-type cells, and no revertant in HAT selective medium containing hypoxanthine, aminopterin, and thymidine. Raji-TG cells, which were maintained routinely in regular medium lacking TG for as long as 2 years, still retained resistance to the drug and inability to grow in HAT medium. A fusion of Raji-TG cells and mouse cells resistant to 5-bromodeoxyuridine and lacking thymidine kinase formed hybrids, and the resulting hybrid colonies proliferated in HAT medium. These observations strongly supported the hypothesis that Raji-TG line cells might be originated from a mutational event with deficiency of HPRT. Both parental and the mutant have a modal chromosome number of 49 with a remarkably stable karyotype. Excess chromosome materials are found in chromosomes 1, 5, 7, 14, and 16. Chromosome 8 is completely missing, but is represented by two respective isochromosomes of the short and long arms of No. 8. Five different marker chromosomes could be distinguished, and most of their origin has been determined. Isolation of Raji-TG X mouse hybrid clones which contained one of each marker chromosome is of considerable value in mapping human genes on regions within particular chromosomes.     

Conformation of amylose in water. I. Light-scattering and sedimentation-equilibrium measurements

The conformation of amylose in aqueous solution has been found to be dependent on its molecular weight. When the molecular weight of amylose is outside of the so-called “dissolving gap” described by Burchard (6500<M_r<160,000) it behaves as a random coil, whereas when its molecular weight is within the “dissolving gap,” it easily aggregates forming a rigid coil which is the B-type (retrograded) amylose. The conformation of this rigid coil is suggested to be a double helix.     

Tissue culture of human epithelial cells from benign colonic tumors

Summary Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for 8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture dish. Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular, was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank carcinoma. This study was supported by NCI Contract N01-CP43366 to M. L. and NCI Grant 1-R26-CA 28822 to E. F.