Utilization of Anhydrous Aluminum Phosphate as a Sole Source of Phosphorous by a Selected Carrot Cell Line

Two variants of carrot (Daucus carota L. cv. MS Yonsun) celllines, which had been selected with Al-phosphate as a sole sourceof phosphorous, were characterized on their mechanisms of phosphate-utilizationfrom Al-phosphate. Both cell lines excreted citrate into themedium. The amount of citrate excretion was highly correlatedwith cell growth in the presence of Al-phosphate. There wasabout a 1 to 1 correlation between solublized-Al and excreted-citratein the medium during cell growth. These results suggest that1) the citrate could chelate with Al, at a 1 to 1 ratio, inAl-phosphate, 2) the citrate-chelated Al remains outside thecells, and 3) solublized phosphate from Al-phosphate is utilizedfor the growth of carrot cells. The characteristics of the selectedcells were very stable, since the rate of citrate-excretionshowed no change after subculturing 25 passages without Al-phosphate. (Received August 7, 1989; Accepted October 19, 1989)     

Expression of NADH-Dependent Glutamate Synthase in Response to the Supply of Nitrogen in Rice Cells in Suspension Culture

The effects of inorganic and organic nitrogen on the levelsof mRNA for NADH-dependent glutamate synthase (GOGAT) and theprotein were examined in rice cells in suspension culture. Asupply of NH⁺₄, NO^-₃, glutamine, or asparagine induced the accumulationof the protein and mRNA, but levels of mRNA for ferredoxin-GOGATwere hardly affected. ¹Present address: P.C. Center Wakuya-cho, Toda-gun, Miyagi,Japan.     

Direct and indirect effects of recombinant human granulocyte-colony stimulating factor on in vitro colony formation of human bladder cancer cells

Although the present experimental use of recombinant human granulocyte-colony-stimulating factor (rG-CSF) has been proven to alleviate the myelosuppression induced by antitumor chemotherapy, it is also believed to stimulate growth of some nonhematopoietic tumor cells. We investigated both the direct and indirect effects of rG-CSF on in vitro colony formation of human bladder cancer cell lines using a modified human tumor clonogenic assay. Peripheral blood mononuclear cells (PBMC) were used as feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish obtained from healthy donors). Human bladder cancer cell lines KK-47, TCCSUP and T24, all derived from human transitional-cell carcinomas, were incubated continuously with various concentrations of rG-CSF ranging from 0.01 ng/ml to 10 ng/ml both with and without PBMC for 7–21 days. The concentrations of rG-CSF used were chosen as being in the range of achievable serum concentrations in patients treated with rG-CSF. At the end of incubation, colonies were counted under an inverted phase-contrast microscope, and an increase in the number of colonies in comparison with the control was used to evaluate the effects of rG-CSF. Results were expressed as a percentage of controls. rG-CSF in the upper layer at concentrations ranging from 0.1 ng/ml to 10 ng/ml stimulated the colony formation of all the cancer cell lines tested in the absence of PBMC in the feeder layer, whereas cells with PBMC in the feeder layer were significantly stimulated more than those without PBMC in the feeder layer (P<0.05) up to a certain concentration, which varied from cell line to cell line. At higher concentrations of rG-CSF, no further stimulation but, on the contrary, a decrease in colony formation was observed in cells with PBMC in the feeder layer in all the cell lines tested. Colony formation in KK-47 and T24 cell lines was significantly inhibited at 5 ng/ml and/or 10 ng/ml rG-CSF compared with cells without PBMC in the feeder layer. Our results suggest that rG-CSF may have both direct and indirect stimulatory effects on the growth of human bladder cancer cell lines in vitro. The results obtained also raise the possibility of adverse effects of rG-CSF in bladder cancer patients whose malignant cells may be directly and indirectly stimulated by this factor while it is being used clinically to alleviate the myelosuppression induced by antitumor chemotherapy.     

Serum-free culture of rat keratinocytes

Summary Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.     

Partial characterization of the glucose transport activity in the golgi-rich fraction of fat cells

The glucose transport activity solubilized from the basal and plus insulin forms of the Golgi-rich fraction of adipocytes was partially characterized, and the results were compared with those of the activity obtained from the plus insulin form of the plasma membrane-rich fraction. The transport activity was determined in a cell-free, reconstituted, system. Prior to reconstitution, the activities in the three preparations were all (a) stable at 0°C for at least 4 h, but not at 37°C or above; (b) most stable at pH 7–9, and (c) less stable in Tes than in Tris buffer. After reconstitution, the three activities were all (d) stable at 0°C, (e) most active at pH 5.5, (f) mildly stimulated by divalent cations, (g) unaffected by insulin or 1 mM of several SH-blocking agents, (h) inhibited by heavy metal ions, 10–100 mM of monovalent salts, organic solvents, several sugar isomers, and specific sugar-transport inhibitors. The rates of d-glucose uptake by the three liposome preparations were all inhibited more strongly by 2-deoxy-d-glucose or $\text{3}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ than by d-glucose. These data indicate that the general properties of the glucose transport activity in the Golgi-rich fraction are similar to those of the activity in the plasma membrane-rich fraction.     

Modification of a novel angiogenic peptide, AG30, for the development of novel therapeutic agents

We previously identified a novel angiogenic peptide, AG30, with antibacterial effects that could serve as a foundation molecule for the design of wound-healing drugs. Toward clinical application, in this study we have developed a modified version of the AG30 peptide characterized by improved antibacterial and angiogenic action, thus establishing a lead compound for a feasibility study. Because AG30 has an α-helix structure with a number of hydrophobic and cationic amino acids, we designed a modified AG30 peptide by replacing several of the amino acids. The replacement of cationic amino acids (yielding a new molecule, AG30/5C), but not hydrophobic amino acids, increased both the angiogenic and the antimicrobial properties of the peptide. AG30/5C was also effective against methicillin-resistant Staphylococcus aureus (MRSA) and antibiotic-resistant Pseudomonas aeruginosa. In a diabetic mouse wound-healing model, the topical application of AG30/5C accelerated wound healing with increased angiogenesis and attenuated MRSA infection. To facilitate the eventual clinical investigation/application of these compounds, we developed a large-scale procedure for the synthesis of AG30/5C that employed the conventional solution method and met Good Manufacturing Practice guidelines. In the evaluation of stability of this peptide in saline solution, RP-HPLC analysis revealed that AG30/5C was fairly stable under 5°C for 12 months. Therefore, we propose the use of AG30/5C as a wound-healing drug with antibacterial and angiogenic actions.     

Factors contributing to cerebral hypomyelination in the growth hormone-deficientlittle mouse

We attempted to delineate the events leading to hypomyelination in the brain of thelittle mouse, a promising murine model of isolated growth hormone deficiency. At 20 days of age, the mutant mouse brain weighed less than its normal counterpart, and this difference in brain weight persisted. Increase in CNPase activity was found to be suppressed in the cerebrum throughout the developmental stage, but not in the other parts of the brain. Differences in cerebral DNA content between thelittle and normal mice first became apparent on the 10th day of age. Thereafter, the rate of increase in thelittle brain consistently lagged behind the normal. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the normal cerebrum is most active, was approximately half that of the controls in all parts of thelittle brain. These findings indicate that the hypomyelination of the mutant cerebrum might result from reduced oligodendroglial proliferation due to growth hormone deficiency.