Phosphorylation of five histone H1 subtypes of L5178Y cells at the exponential growth and mitotic phases

Histone H1 of cells of L5178Y, a mouse lympholeukemic cell line, consists of five molecular species designated as H1-I, II, III, IV, and V. The phosphorylation of these H1 subtypes was examined at the exponential growth phase and during mitosis, by BioRex 70 column chromatography and two-dimensional polyacrylamide gel electrophoresis. In exponentially growing cells, the degree of phosphorylation was different for each subtype. H1-II was the most highly phosphorylated, 1.8 phosphate residues per molecule, followed by H1-IV/V, 1.4, I, 0.8, and III, 0.5. In the mitotic phase, H1-II was also the most highly phosphorylated 6.0 phosphate residues per molecule, H1-IV/V, 3.5, I, 2.7, and III, 1.2. The phosphorylation started simultaneously among the subtypes after colcemid addition, and phosphorylated H1 subtypes accumulated linearly. The rate of incorporation of 32P into each H1 subtype was almost constant during colcemid treatment. During 4 h after colcemid addition, the phosphate residues incorporated into H1 did not dephosphorylated. The H1 kinase activities increased to six times higher during the colcemid treatment.     

Photochemical inactivation of human placental estradiol 17 beta-dehydrogenase in the presence of 2,3-butanedione

Estradiol 17 beta-dehydrogenase of human placenta was rapidly inactivated by 2,3-butanedione under u.v. light, and no protection against the inactivation was observed in the presence of sodium azide. Under ordinary laboratory illumination, the inactivation was biphasically progressed in time-dependent and concentration-dependent manners, while a partial protection from the inactivation was indicated by sodium azide. These results suggest that the inactivation mechanism of the dehydrogenase by 2,3-butanedione under laboratory illumination is different from that under u.v. light. Therefore, the inactivation under laboratory illumination proceeded by a reaction with excited singlet molecular oxygen (1 delta g or 1 sigma +g states), and that under u.v. light was caused by a reaction of substrate with triplet sensitizer. In the presence of NADP+, the inactivation of the enzyme by 2,3-butanedione was markedly reduced. The maximum protection by NADP+ was about 80% of the initial enzyme activity. Amino acid analysis of the enzyme treated with 2,3-butanedione under laboratory illumination showed that the modified enzyme contained considerably less of the following amino acids than the native enzyme: histidine, arginine, threonine, methionine, tyrosine and leucine. In addition, other dicarbonyl reagents, 1,4-dibromo-2,3-butanedione, 1-phenyl-1,2-propanedione, phenylglyoxal, 16-oxoestrone, 1,2-cyclohexanedione, 2,4-pentanedione and glyoxal were found to decrease the dehydrogenase activity in various degree.     

The 25-kilodalton hemolytic protein affinity-purified from parasporal inclusions ofBacillus thuringiensis strain PG-14 (serotype 8a∶8b)

A simple method using an antibody-mediated affinity chromatography was developed for rapid and specific purification of the 25-kilodalton protein from alkali-solubilized and silkworm (Bombyx mori) larval gut juice-digested parasporal inclusions of theBacillus thuringiensis strain PG-14 (serotype 8a8b). Affinity-purified 25-kilodalton protein was highly hemolytic to red blood cells (RBCs) of two avian (chicken and goose) and six mammalian (horse, mouse, cow, rabbit, guinea pig, and sheep) species. The concentration of the 25-kolodalton protein required for 100% hemolysis was in the range of 2–16 g/ml, and an apparent RBC species-dependent variation was observed in hemolytic activity of this protein. Of the RBCs tested, chicken and house RBCs were the most susceptible to hemolysis by this protein; sheep RBCs wre 4–8 times less susceptible than the others.     

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)     

Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica)

We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5 and 3 ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG) _n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT) _n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.Paper 3082 of the Forest Research Laboratory, Oregon State University     

Automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography

An automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography (HPLC) with column switching is described. The system consists of a reversed-phase column, a cation-exchange column, a column switch, four sets of ultraviolet absorbance detectors, a microcomputer and other conventional equipment. As this system permits the simultaneous determination of urinary orotic acid, uracil, dihydrouracil, pseudouridine, xanthine, 2,8-dihydroxyadenine and succinyladenosine, it offers a useful method for the detection of orotic aciduria, dihydropyrimidine dehydrogenase deficiency, diphydropyrimidinuria, xanthinuria, adenine phosphoribosyltransferase deficiency and adenylosuccinase deficiency.     

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.