The R-banding pattern of the Chinese hamster Don cell line

Chinese hamster cells (Don line) were treated in vivo with 5-BrdU and 33258-Hoechst fluorochrome for obtaining the partial inhibition of condensation that causes the R-banding pattern. Untreated chromosomes were stained by a standard G-banding method. Statistical measurements show significant differences in the band numbers between the two treatments. The Don cell line in the authors' laboratory presents some karyotypical differences from Don cell lines studied by other authors.     

Sensitivity of two Chinese hamster cell lines to SCE induction by a variety of chemical mutagens

SCE induction in Chinese hamster Don (lung) cells was compared with that in CHO (ovary) cells exposed under identical conditions to 14 known mutagens. Test protocols used for comparison were selected following a study of Don and CHO cell responses to aflatoxin B1 and benzo[a]pyrene. In the absence of added metabolizing enzymes 9-aminoacridine, 4-nitroquinoline 1-oxide, N-methyl-N-nitrosourea, dimethylcarbamoyl chloride, beta-propiolactone, daunomycin, aflatoxin B1 and 2-aminoanthracene were directly active in both cell lines; every substance positive in CHO cells was also positive in Don cells. However, the latter detected cyclophosphamide, hydrazine sulphate, benz[c]acridine, 3-methylcholanthrene and benzo[a]pyrene without addition of S9. CHO cells did not respond equivalently to these mutagens, either in the presence or absence of S9. Other differences between the cell lines depended on chemical exposure time, S9 pre-incubation or co-incubation conditions. For example, the ability of CHO cells to detect SCEs due to 2-aminoanthracene was acutely dependent on exposure time. In addition, Don cells exhibited lower background SCE values which were less variable than those of CHO cells under the same culture conditions. Although incapable of detecting 4-dimethylaminoazobenzene (butter yellow) and not as sensitive to cyclophosphamide as certain cell lines of liver origin, the pseudodiploid Don cell line possesses other desirable characteristics required for in vitro SCE assays, particularly with regard to intrinsic metabolic activation of polycyclic aromatic hydrocarbons and related substances.     

Evolution of digestibility by Hind III: an analysis by light and electron microscopy

Digestion of Chinese hamster metaphase chromosomes from the Don cell line by Hind III restriction endonuclease followed by Giemsa staining were analysed by light and electron microscopy. The evolution of digestibility was studied and four digestion stages were characterized by different levels of chromosome structure. Three different condensation stages were established according to morphological criteria of length, width and separation among chromatids. It was observed that there are statistically significant differences in the digestion progress at the three condensation stages previously defined.     

Evolution of digestibility by Hind III: an analysis by light and electron microscopy

Digestion of Chinese hamster metaphase chromosomes from the Don cell line by Hind III restriction endonuclease followed by Giemsa staining were analysed by light and electron microscopy. The evolution of digestibility was studied and four digestion stages were characterized by different levels of chromosome structure. Three different condensation stages were established according to morphological criteria of length, width and separation among chromatids. It was observed that there are statistically significant differences in the digestion progress at the three condensation stages previously defined.     

The chinese hamster Don cell line differs from normal diploidy by one chromosome band

G-banded and C-banded prometaphase and metaphase karyotypes of the Chinese hamster Don cell line from three different laboratories were compared. All the cell lines were pseudodiploid. Each consisted of two distinct cell populations designated S1 and S2. The chromosome complements of each of these populations differed from the diploid by only one additional chromosome band on a subterminal 1p in S1 and on a median 1q in S2. The sites of these extra bands were neither constricted nor heterochromatic. All other different karyotypes, including those from seven distinct subpopulations, had basic patterns of chromosome complements that were best represented by S1 or S2, respectively the major or minor cell type of the cell line. Moreover, nearly 80% of the metaphases analyzed had a modal chromosome number of 22, and about 60% had the same chromosome composition of a cell type or subtype. Our data also suggest that Don cells were probably pseudodiploid by 1964 or before.     

Asymmetric protoplast fusion between sweet potato and its relatives,and plant regeneration

Experiments were conducted to asymmetrically fuse protoplasts from sweet potato (Ipomoea batatas L. Lam.) and its wild relativesI. trifida Don. andI. lacunosa L. Protoplasts of sweet potato were treated with iodoacetamide, whereas those ofI. trifida Don. andI. lacunosa L. were irradiated with X-rays. The asymmetric protoplast fusion was carried out by the electrofusion method and by polyethylene glycol treatment. Electrically-fused protoplasts initiated cell division, and then formed calli earlier than the polyethylene glycol-fused protoplasts. Plant regeneration occurred only in electrofused calli, suggesting that polyethylene glycol had some toxic effect on plant regeneration ability. Analysis of peroxidase isozymes confirmed the interspecific hybrid characteristics of both the fusion-derived calli and regenerated plants.     

Flora Palinologica Italiana,Sezione Aeropalinologica-Specie esotiche S229:Thuja orientalis L. (Cupressaceae); S230:Cryptomeria japonica (L. fil.) D. Don (Taxodiaceae)

Summary The morphopalynological cards ofThuja orientalis L. and ofCryptomeria japonica (L. fil.) D. Don were illustrated as part of a more comprehensive study on ornamental Pinophyta. Pollens from the original area of the two species were also compared with grains coming from plants cultivated in Italy. In the case ofCryptomeria japonica (L. fil.) D. Don DV, D01, D02, exine, min. and max. diameters, calculated on a japanese sample, differed significatively from the italian sample. In the case ofThuja orientalis L. no significant differences were observed. Pollen from the two italian sites did not show significative differences in size and shape. Pollens acetolysed with the traditional method (Erdtman, 1960) were compared with pollens treated with the Tatzreiter method (1985). Morphometric analysis showed similar values for both species.     

The isolation and characterization of asparagine-requiring mutants of Chinese hamster cells

Nine asparagine-requiring mutants were isolated in culture from the Don line of Chinese hamster cells. Investigation of the asparagine requirements of the mutants, the effect of asparagine deprivation on macromolecular synthesis, and the rates of reversion to asparagine independence indicated that there were differences between the mutant clones. Biochemical analysis revealed that the defect in the mutants was due to a deficiency of the enzyme asparagine synthetase, and that the enzyme activity in the mutants and Asn⁺ revertants obtained from them was not influenced by the concentration of asparagine in the growth medium. Complementation analysis by Sendai virusmediated cell fusion indicated that the lesion behaved as a recessive trait, and was probably located in the same gene in all the mutant clones.     

Concanavalin A induces endoreduplication in cultured mammalian cells.

Concanavalin A (Con A) induced endoreduplication in an established cell line, Don, of the Chinese hamster. The inducibility of Con A was inhibited by α-methyl-D-mannoside. When a secondary culture of kidney cells (CHK), which showed the contact-inhibition of growth, was used, there was an increase in spontaneous endoreduplication. CHK cells or some of them were more sensitive to Con A than Don cells, in which few spontaneous endoreduplications were observed. Mitotic shake-off after Con A treatment led to the higher ratio of endoreduplicated cells to normal mitoses, suggesting that endoreduplicating cells do not “round-up” and probably do not condense chromosomes through the cell cycle until M is reached.     

20-hydroxyecdysone accumulation and regulation in Ajuga lobata D. Don suspension culture

Suspension culture of Ajuga lobata D. Don cells provides a method of synthesis of the phytoecdysteroid 20-hydroxyecdysone (20E) which can regulate the molting process of larvae. We characterized the culture conditions to optimize 20E production. Growth of A. lobata D. Don cells fits the logistic equation curve with a growth cycle of 19 days. Medium conductivity was negatively correlated with dry cell weight and 20E accumulation, thus could be used to determine the optimal time for cell harvest. Continuous subculture reduced 20E synthesis, but supplementing medium with 20E precursors mevalonic (MVA), α-Pinene, and nitric oxide (NO) can significantly promote cell growth and influence 20E accumulation. Combination of α-Pinene, MVA, and SNP significantly elevated 20E accumulation, thus may synergistically enhance 20E synthesis in A. lobata D. Don. The optimal concentrations of α-Pinene, MVA, and NO donor SNP in suspension culture were 50 μL L^?1, 10 mg L^?1, and 80 μmol L^?1.     

Modulation between aerobic and anaerobic metabolism in the mutant cell line CdtR-Q

It has recently been shown that the cell line Don Q obtained by mutagenesis of wild type Chinese hamster lung fibroblasts (Don wt), presents a point mutation in the gene coding for UDP-glucose pyrophosphorylase. The persistent low level of UDP-glucose makes Don Q clone resistant to Clostridium difficile toxin B. Starting from the observation that Don Q cells exhibit many large hydrophobic cytoplasmic inclusions, that we have found to be made of neutral lipids, the aerobic metabolism of the two cell lines has been examined. The specific activity of cytochrome oxidase in Don Q cells is more than 5 times lower than that found in Don wt. Also, the activity of Complexes II + III, expressed by the activity of succinate-cytochrome c oxido-reductase, has been found to be lower in Don Q compared to wt cells. On the other hand, NADH-cytochrome c oxido-reductase activity, insensitive to rotenone, is more than doubled in Don Q. In these cells the activity of lactate dehydrogenase is very high, being able to oxidise more than 3,000 nmoles of NADH/min/mg of protein. The results obtained indicate that Don Q cells, in addition to a decreased ability to synthesise UDP-glucose, have an impairment in the respiratory chain. Such an impairment could be correlated to the increased capacity to generate a higher amount of reducing equivalents through the glycolytic activity, which can then be utilised for the synthesis of fatty acids stored in lipid droplets.     

Isolation and characterization of intergeneric somatic hybrids in the Apocynaceae family

Summary Protoplasts were isolated from callus cultures of Rauwolfia serpentina Benth., Rhazya stricta Decaisne, and Catharanthus roseus (L.) G. Don, or from leaves of Vinca minor L. Protoplast isolation, culture, and fusion techniques as well as hybrid screening systems were developed for these species, and hybrids were obtained. Hybrid combinations were Rauwolfia + Vinca, Rauwolfia + Catharanthus, Rauwolfia + Rhazya, and Catharanthus + Vinca. For hybrid isolation, the physiological complementation method was utilized. Analyses of the material obtained included a cytogenetic study of the chromosomes, a study of multiple molecular forms of transferases and esterases, and the blot hybridization of restricted nuclear DNA using ribosomal DNA as a probe. Hybrids were identified in all species' combinations tried. A ten-fold increase in the accumulation of raucaffricine (relative to the parental Rauwolfia strain) was observed in one cell line of the Rauwolfia + Vinca hybrid. Our studies indicated the genetic stability of the great majority of the hybrid cell lines over a period of more than 20 months of in vitro growth. No shoot morphogenesis has so far been observed in this material.     

Assignment of the gene for cystathionine β-synthase to human chromosome 21 in somatic cell hybrids

Summary Among several established mouse, rat, and Chinese hamster cell lines that were screened for cystathionine -synthase (CBS) activity, mouse 3T3 and Chinese hamster Don fibroblasts were found to contain no detectable activity. Somatic cell hybrids between human fibroblasts KG-7 with normal CBS activity and Don/a23TK^- cells (series XXI) were examined for CBS activity and for human chromosome content. Only chromosome 21 cosegregated with CBS activity. Because the activities measured could represent either Chinese hamster or human gene products, we have prepared a new series of hybrids between Don/a23TK^- cells and mutant human fibroblasts from a patient with homocystinuria due to deficiency of functional CBS mRNA. None of these (series XXV) hybrids contained detectable CBS activity, although collectively all human chromosomes were represented. Our results suggest that the human gene for CBS, called CBS, and thus for the most common form of homocystinuria, is located on chromosome 21.     

Single-cell cloning of a crown gall from protoplasts regenerated in hormone-free medium. Establishment of pure transformed cell-lines of Catharanthus roseus

Protoplasts enzymatically isolated from cell line of Catharanthus roseus G. Don crown gall, were cultured at high density (10⁵ P ml^-1) in modified B5 liquid medium (Gamborg et al. 1976). In the absence of growth regulators C. roseus protoplasts were able to regenerate a cell-wall, divide and, subsequently, yield very numerous clones in the absence of growth regulators. After two weeks, the cultures were greatly diluted in order to obtain clones of single-cell origin. Most of the clones individually transferred onto solid medium can proliferate indefinitely, without growth regulators. Among analyzed clones, 90% were nopaline positive. Their ajmalicine and serpentine content was compared with that of the parental crown gall line, and was found to be low. The CR10 protoplasts were very easy to grow, they were an interesting model for the development of pure tumorous lines. Moreover, we found that the tumorous protoplasts were useful for cell fusion experiments or for the delicate culture of tree protoplasts.Abbreviations B5 Gamborg et al. (1976) medium - 2,4-d 2,4-dichlorophenoxyacetic acid - Kin Kinetin - NAA naphthalene acetic acid - BA N⁶ (benzyl) adenine     

Observations of mitochondria and mitochondrial nuclei by double staining with rhodamine-123 and DAPI in synchronous cultures ofCatharanthus roseus

Mitochondria in cells ofCatharanthus roseus (L.) G. Don in synchronous cell division cultures were observed by double staining using fluorescence microscopy. The cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) first and subsequently stained with rhodamine 123 (r-123). Immediately after staining with r-123, yellowishgreen, elongated and moving mitochondria were observed upon excitation at 485 nm. When the excitation filters were replaced by a UV filter (360 nm), 1 to 7 mitochondrial nucleoids were visible in each mitochondrion in the same field. Changes in the lengths of mitochondria during the cell cycle obtained from the observations under fluorescence microscopy by this staining method suggest the occurrence of multiplication of mitochondria concurrent with the cell cycle ofC. roseus.     

Telomere erosion-induced mitotic catastrophe in continuously grown chinese hamster don cells

We have previously reported that telomere erosion is the earliest chromatin modification in cells entering the apoptotic pathway. The purpose of this investigation was to determine whether loss of telomeric DNA was involved in inducing mitotic catastrophe and death in Chinese hamster Don cells. Don, a male Chinese hamster-derived cell line which requires daily subculturing to remain diploid, was grown without subculturing for 1-4 days at 37 degrees C and analyzed cytologically. Our results indicated that (1) the frequency of metaphase chromosomes with structural anomalies was significantly higher in 3-day continuously grown cells than in 1-day control cells (8.2% vs 5.7%; P < 0.01), (2) the mitotic index was considerably lower in 3-day continuously grown cells (0.13%) than in control cells (3.64%), (3) cells grown for 3 days continuously showed a higher incidence (7.6%) of endoreduplicated metaphase chromosomes than did control cells (4.9%), (4) 4-day continuously grown Don cells showed significantly smaller amounts of telomeric DNA in interphase nuclei than did control cells, and (5) apoptotic cells were more frequent in 4-day cell cultures (40.6%) than in control cells (4.3%). These results support our earlier observations and contribute additional support for our hypothesis that telomere reduction is the cause of mitotic catastrophe and that cell death in continuously grown Don cells occurs because of the loss of telomeric DNA.     

Insect-resistant transgenic Pinus radiata

Transgenic radiata pine (Pinus radiata D. Don) plants containing a Bacillus thuringiensis (Bt) toxin gene, crylAc, were produced by means of biolistic transformation of embryogenic tissue. Using the selectable marker gene nptII and corresponding geneticin selection, 20 independent transgenic lines from five genotypes were established. Over 200 plants regenerated from ten transgenic lines were successfully transferred to soil. The integration and expression of the introduced genes in transgenic tissue and/or plants were confirmed by PCR, Southern hybridisation and neomycin phosphotransferase II (NPTII) and Bt ELISA assays. Bioassays with larvae of the painted apple moth, Teia anartoides, demonstrated that transgenic plants displayed variable levels of resistance to insect damage, with one transgenic line being highly resistant to feeding damage.     

Line thinning enhances diversity of Coleoptera in overstocked Cryptomeria japonica plantations in central Japan

We evaluated the effectiveness of line thinning, a new silvicultural technique, toward restoring diversity of Coleoptera in overstocked Cryptomeria japonica D. Don plantations in central Japan. We compared the abundance of some common Coleoptera families between line-thinned stands and adjacent unthinned stands in two plantations: low-elevation Sugi site (4 years since thinning) and high-elevation Kuchiotani site (6 years since thinning). Many bettle families comprising various functional groups such as plant feeders, wood borers, rotten wood feeders, root feeders, fungus feeders, dung feeders, and scavengers were more abundant in the line-thinned stands than in the unthinned stands. Furthermore, some important families were missing from the unthinned stands. There were strong positive relationships between Coleopteran abundance and understory vegetation. Our results suggest that line thinning may potentially increase biodiversity in overstocked C. japonica plantations by restoring important ecological processes such as food-web interactions (pollination, predation, herbivory, decomposition, parasitism, etc.), and habitat conditions.     

Bioconversion of 3-chlorobenzoate to 2-chloromuconate controlled by on line HPLC

Summary Strain RD330 a transposon mutant of Alcaligenes eutrophus JMP134 was considered to be dienelactone hydrolase defective (Don et al. 1985). During a bioconversion experiment with 3CB (3-chlorobenzoate) 2CMA (2-chloro-cis,cis-muconate) was accumulated by RD330 with an overall amount of 31%, but no dienelactone could be detected. Enzyme tests revealed that both enzymes 2CMA-cycloisomerase and dienelactone-hydrolase were induced at low levels in RD330 by 3CB and its metabolites.The control of 3CB addition during the bioconversion experiment was performed by on line HPLC (high pressure liquid chromatography).