Caffeic acid-O-methyltransferase in a suspension of cell aggregates of tobacco

Aggregates of tobacco cells in suspension in 2,4-D (10^?6 M) and kinetin (10^?5 M) cultures were fractionated by size, then their O-methyltransferase (OMT) activities were assayed. Only the kinetin culture showed high OMT activity, which was higher in the larger than the smaller aggregates at all stages of cell growth. The contents of phenolic acids were also greater in the larger cell aggregates in the kinetin culture. However, when the kinetin cultured cells were transferred to a medium containing 10^?6 M of 2,4-D, the relationships between the cell size of the aggregates and OMT, lignin and the phenolic acids disappeared. The importance of kinetin and cell association for OMT and the subsequent lignification of the cells is discussed.     

The Effect of Exogenous IAA and Kinetin on Nitrate Reductase,Nitrite Reductase and Glutamate Dehydrogenase Activities in Excised Pea Roots

Nitrate reductase (NO₃R) activity, nitrite reductase (NO₂R) activity and NADH₂ dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10^?5; 3 × 10^?5; 5 × 10^?5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1^?1, that is 4.65 × 10^?7;4.65 × 10^?6 and 2.3 × 10^?5 M). NO₃R activity was not influenced by IAA, NO₂R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO₃R activity significantly both after 9 h and 24 h incubation, slightly increased NO₂R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO₂R activity. Nevertheless the depressing effect of kinetin on NO₃R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.     

A tissue culture of the Ostrich fern Matteuccia struthiopteris (L.) Todaro

Multiple bud formation was induced from shoot apices of Matteuccia struthiopteris cultured on semi-solid Knudson's medium supplemented with 10^-5 and 10^-6 M kinetin. The effect of kinetin, naphthaleneacetic acid and gibberellic acid on shoot and root development is discussed and a three-part tissue culture system was devised for micropropagation and rooting.     

Histidase function in human epidermal cells

The activity of histidase was studied in (1) epidermal tissue scraped from human infant foreskin, (2) fibroblast-like cells in monolayer serial culture from human foreskin, and (3) epithelial-like (epidermal) outgrowth from foreskin primary explants. Foreskin epidermal tissue without in vitro culture and epidermal outgrowth in primary culture from explants of foreskin showed equivalent mean levels of histidase activity, 5.22 × 10^?3 and 5.01 × 10^?3 μMoles urocanic acid produced per milligram protein per minute. Under the same assay conditions, there was no measurable histidase activity in cultured fibroblast-like cells from foreskin at various times after subculture. The K_m for enzyme from human foreskin epidermal tissue ranged between 2 and 5 × 10^?3 M histidine. Ability to demonstrate the presence or absence of this tissue-specific enzyme function in cultured cells suggests a useful means for studying differentiation, as well as a more precise way to identify epidermal origin of cultured cell types than morphological characteristics alone would permit.     

Origin of thymidine kinase deficient (TK-) haploid frog cells via an intermediate thymidine transport deficient (TT-) phenotype

Wild-type cultured cells of the frog cell line ICR 2A give rise to 5-bromodeoxyridine (BUdR)-resistant colonies only when the selecting concentration of the drug is 5 × 10^?5 M or lower. The progeny of these colonies multiply in 10^?4 M BUdR; resistance is correlated with the absence of a thymidine (TdR)-specific transport reaction with a K^m in the range of 2–7 × 10^?4 M. All of the TdR transport-deficient (TT-) isolates examined (25) had TdR kinase activity (4% to 100% of wild-type). Variants deficient in TdR kinase activity (5% of wild-type) were obtained by exposing TT-cultures to 10^?3 M BUdR. The TK - variants multply continuously in 10^?3 M BUdR and retain the phenotype after prolonged culture in the absence of the drug. The frequency with which they occur is increased 20 to 50 fold by prior treatment of the culture with ICR 191, an acridine mustard mutagen. In haploid cells, it would be expected that TK- variants would arise in equal numbers from wild-type and TT- cultures if loss TdR kinase occurred independently of loss of the transport reaction. However, wild-type cells give no colonies resistant to 10^?3 M BUdR under conditions the give 1 to 50 colonies per million TT- cells. The TT- phenotype seems to be a required intermediate state in the origin of the TK- phenotype. Therefore, the TK- clones described above are unlikely to be products of mutation at a single genetic locus.     

Cytogerontological studies of biological activity of oregano essential oil

In order to clarify possible cytological mechanisms that underlie the beneficial effects of carvacrol-bearing essential oils on health and mental abilities, we studied one of them (oregano essential oil) in experiments on transformed cultured Chinese hamster cells. Possible cytotoxic or mitogenic effects of the preparation at various concentrations were preliminarily estimated by analyzing the cell culture density after 4 days of cultivation. The preparation concentration in the growth medium (on carvacrol basis) varied from 1 × 10^?15 up to 5 × 10^?4 M (on carvacrol basis). As a result, two concentrations were selected for further experiments, including 2.5 × 10^?5 M as the maximal absolutely non-toxic concentration and 2.5 × 10^?4 M as the concentration at which the oregano essential oil decreased approximately 2-fold the final cell density of the grown culture. It was found that the preparation at 2.5 × 10^?5 M had no effect on either the colony-forming ability of the cells or the saturation density of the culture (which is a marker of its ??biological age??) or kinetics of its ??stationary phase aging?? (degradation of cultured cells in the stationary phase of growth, similar to age-related changes of the cells in aging organism). On the contrary, the oregano essential oil at 2.5 × 10^?4 M abruptly diminished colony-forming ability of the cells and influenced as a ??pro-aging?? factor on the saturation density of the cell culture and kinetics of the cell death induced by ??stationary phase aging.?? Based on our own concept of aging and the data obtained, we assumed that detected increase in the life span of mice under the influence of the oregano essential oil could be determined by certain functional changes at the organismal level only, but is not associated with any geroprotective (anti-aging) activity of the preparation, which is manifested at the cellular level and improves the cell viability.     

Kinetin inhibition of h-uracil and C-leucine incorporation by tobacco cells in suspension culture

The rate of ¹⁴C-leucine and ³H-uracil incorporation by tobacco cells (Nicotiana tabaccum var. Samsun N.N.) in suspension culture was simultaneously decreased by the addition of kinetin at concentrations above 2.5 × 10⁻⁵m. Ribosomal RNA was the first RNA species affected by kinetin. The purine derivatives, adenine and N⁶-methyl-aminopurine, which exhibit low cytokinin activity overcame the inhibitory effects of kinetin. However, purine derivatives without cytokinin activity, guanine, N^6,6-dimethyl-aminopurine, and 2-aminopurine, did not relieve kinetin inhibition.     

In Vitro Regenerative Competence of Cleisostoma racimeferum (Orchidaceae) Aerial Roots

Regeneration competence of aerial roots of Cleisostoma raeimeferum (Orchidaceae) from in vivo and in vitro sources was tested. The protocorm-Iike bodies and shoot buds were obtained from 2 w old in vivo grown aerial roots and 20 wold in vitro grown roots on Murashige and Skoog medium containing sucrose (3%) (w/v), casein-hydrolysate (2 g l^?1), coconut water (15%) (v/v), citric acid (200 mg l^?1) and different plant growth regulators. The morphogenetic response from in vivo grown roots was poor and only 20% of the cultures yielded protocorm-like bodies and shoot buds on medium containing IAA (2 µM) and kinetin (2 µM) in combination after 75 d of culture. While 100% morphogenetic response was exhibited by in vitro grown roots on MS medium enriched with IAA (1 µM) and kinetin (1 µM) in combination only after 25 d of culture initiation. The response initiated at the cut ends of the roots and subsequently the entire root length was taken over. Both IAA and kinetin singly stimulated mostly callusing of the explants. The rooted plantlets and multiple shoot buds were obtained after 30 d of culture from protocorm-like bodies and shoot buds on basal medium enriched with IAA (2 µM) and kinetin (6 µM) in combination. The well developed rooted plants could be obtained for transferring to potting mix after ～24 w of culture initiation.     

Activation and inhibition of the action potential Na+ ionophore of cultured rat muscle cells by neurotoxins.

Both myoblasts and myotubes in cultures of clonal rat muscle cells have action potential Na⁺ ionophore activity. The ionophore is activated by batrachotoxin (K_0.5 = 3 to 5 × 10^?7 M) and veratridine (K_0.5 = 4 to 6 × 10^?5 M) which compete for the same activation site. As in denervated rat muscle, the ionophore of cultured muscle is 100 fold more resistant to inhibition by tetrodotoxin (K_0.5 = 1.5 to 3 × 10^?6 M) and 20 fold more resistant to inhibition by saxitoxin (K_0.5 = 1.5 to 3 × 10^?7 M) than in nerve, innervated muscle, or cultured neuroblastoma cells.     

Notes on the Taxonomy of the Genus Philonotis by means of Vegetative Characters

Abstract

Three cytokinins tested, BAP, 2iP and kinetin at concentrations of 10^?8 – 10^?4 M, induced buds on Jhe protonema of Bryum pallescens, which otherwise remains bud-free on basal medium in ordinary cultural conditions. BAP proved best for bud induction, and was followed in effectiveness by 2iP and kinetin. Protonemal growth decreased with increase in concentrations of BAP, but with 2iP and kinetin it increased only with increase in concentration up to 10^?5 M.     

Hormonal control of deoxyribonucleic Acid and protein syntheses in pea root cortical explants

The hormonal control of DNA and protein syntheses in cortical explants taken at 10 to 11 mm from the tip of 3-day-old seedling roots (Pisum sativum cv. Little Marvel) was examined. On the auxin medium, S2M, the cortical cells began to enlarge at day 4 in culture, with no DNA synthesis or cell division throughout the 7-day culture period. With the addition of kinetin to this medium, S2M + K, the DNA content of the explants increased about three times by day 3, with further increases thereafter. This DNA increase was followed by cell division activity and subsequent tracheary element differentiation initiated at day 5. At least two divisions per parent cortical cell were required prior to this cytodifferentiation. The absolute hormonal requirements for the DNA synthesis and cell division responses were substantiated by the lack of either response in explants cultured on basal (S2M medium minus auxins) or basal + K medium for 7 days. On the auxin medium, there was no protein accumulation in the cortical explants over the 7-day period. On S2M + K medium, protein accumulation began after day 2 with a steady rate of increase until day 4, and some fluctuation thereafter. The pattern of increasing uptake of ¹⁴C-leucine was similar for days 0 to 4 in explants on either medium. After day 4 on S2M, the uptake continued to increase coincident with cell enlargement initiation, whereas on S2M + K there was a decline. Incorporation of ¹⁴C-leucine into trichloroacetic acid-precipitates of the total buffered homogenate from explants on both media exhibited a similar pattern, i.e. an increase during days 0 to 3 and then a decline to a level about three times higher than day 0. Incorporation into the homogenate soluble fraction also showed a similar pattern in explants cultured with or without kinetin. From the differences in net protein accumulation and the incorporation data, speculation on a cytokinin effect on protein synthesis and degradation rates is presented.     

The inhibition of the serum-stimulated increase of ornithine decarboxylase by ionophores and local anesthetics

The addition of fresh serum-containing growth medium to L1210 mouse leukemic cells in culture resulted in a 5-fold increase in ornithine decarboxylase (l-ornithine carboxy-lyase, EC 4.1.1.17) activity. The presence of microtubule disrupting agents (colchine, vinblastine) or cations (5–10 mM K⁺, Na⁺ or Mg²⁺) abolishes this increase of ornithine decarboxylase activity (Chen, K.Y., Heller, J.S. and Canellakis, E.S. (1976) Biochem. Biophys. Res. Commun. 70, 212–219). Based on these observations we proposed that fluctuation in cellular cation concentrations may act as a link between the membrane structure and ornithine decarboxylase. To test this proposal, we studied the effects of selective membrane perturbing agents such as ionophores and local anesthetics, on the serum-stimulated increase of ornithine decarboxylase activity in L1210 cells. Among the six inonophores tested, valinomycin was the most potent one, with I₅₀ value (concentration that gives 50% inhibition of orthinine decarbocylase activity) of 6·10^?9 M. Dibucaine and tetracaine were also effective inhibitors at 10^?4?10^?5 M. The I₅₀ values of valinomycin on the protein synthesis and RNA synthesis, however, were greater than 1·10^?6 M. These results substantiate the notion that ornithine decarboxylase activity can be regulated at plasma membrane level and such regulation is related to the perturbation of cellular cation pools.     

The effect of hormones on anthocyanin accumulation in cell cultures of Haplopappus gracilis

Summary Suspension cultures of Haplopappus gracilis accumulated anthocyanin when grown in defined media with 4.5×10^-6M 2,4-D. Transfer of cells to media with 10^-5M kinetin or benzyladenine and no auxin or 10^-7M NAA for 6 days resulted in increased anthocyanin concentration of the cells but the total amount of pigment was unaffected due to differences in growth rates. The cultures yielded up to 35 mg pigment per gram dry weight.Cells grown in batch culture in media with 10^-5M kinetin and with 10^-7 M NAA or 5×10^-5M NAA sampled and analyzed daily grew at the same rate. The concentration of anthocyanin differed, being lower in cells at 5×10^-5M NAA. After 6 days there was a rapid increase in pigment formation, and by 14 days the concentration of anthocyanin in cells in the two media were the same.When the cells were cultured in 3.5-1 phytostats and 600 ml culture was replaced daily with 600 ml medium, anthocyanins accumulated when the NAA concentration was 10^-7M but not at 10^-6M. At 10^-7M NAA the cultures remained pigmented and anthocyanin accumulation could be restored after a temporary loss of pigmentation due to an earlier, higher auxin concentration. The changes in concentration of phenylalanine ammonia-lyase did not correspond to changes in the rate of anthocyanin accumulation. The enzyme showed a maximum 4–8 h after inoculation of cells to fresh media. Cells grown on agar plates and rich in anthocyanin were observed to divide without loss of pigmentation, demonstrating that cells differentiated with respect to anthocyanin production undergo mitosis.Issued as NRCC No. 11388.Abbreviations used: 2,4-D=2,4-dichlorophenoxyacetic acid, NAA + -naphthaleneacetic acid.     

Effect of synthetic auxins on callus induction from tea stem tissue

A study was initiated to establish an in vitro culture protocol for tea (Camellia sinensis). Explant sources, disinfestation methods and culture media were examined. Segments (divots) were dissected from greenwood stem (current year growth) internodes of field grown plants. Disinfestation was achieved by separate treatments of 3.75% sodium hypochlorite and 7.5% CaCl₂. MS medium with sucrose (30 g/L), inositol (100 mg/L) and thiamine-HCl (1.3 mg/L) and kinetin was used with combinations of the auxins: (2,4-dichlorophenoxy) acetic acid (2,4-D), (2,4,5-trichlorophenoxy) acetic acid (2,4,5-T), (naphthalene) acetic acid (NAA) and 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (Picloram). Picloram (10^-7M) induced the most callus proliferation without kinetin. At a constant level of kinetin (10^-5M), the concentrations inducing the most callus growth were 10^-7M for 2,4-D, 10^-6M for 2,4,5-T, 10^-7M for Picloram and 10^-8M for NAA. A factorial test of 2,4,5-T and kinetin concentrations showed the optimum for callus growth was 10^-7M and 10^-5M, respectively.Technical Contribution No. 2532 of the South Carolina Agricultural Experiment Station, Clemson University.Graduate Research Assistant and Professor, respectively.     

Dexamethasone counteracts the anti‐osteoclastic,but not the anti‐leukemic,activity of TNF‐related apoptosis inducing ligand (TRAIL)

We have analyzed the effect of the synthetic glucocorticoid dexamethasone, used alone or in combination with recombinant TRAIL, on in vitro osteoclastic differentiation of peripheral blood‐derived macrophages cultured in the presence of macrophage‐colony stimulating factor (M‐CSF) + RANKL for 12–14 days. Dexamethasone exhibited different effects based on the concentration used. Indeed, while at 10^?7 M dexamethasone reduced the number of mature osteoclasts, at 10^?8 M showed no significant effects and at 10^?9 M significantly increased the number of mature osteoclasts, with respect to cells cultured with only M‐CSF + RANKL. On the other hand, the addition in culture of recombinant TRAIL inhibited the output of mature osteoclasts induced by M‐CSF + RANKL. However, the presence of dexamethasone (10^?8 or 10^?9 M) into the culture medium significantly counteracted the anti‐osteoclastic activity of TRAIL. In order to ascertain whether dexamethasone, might also interfere with the anti‐leukemic activity of TRAIL, the degree of apoptosis induced by TRAIL was evaluated in several myeloid (OCI, MOLM, HL‐60) and lymphoid (SKW6.4, MAVER, BJAB) leukemic cell lines. The levels of TRAIL‐triggered apoptosis were not significantly different between leukemic cells cultured in the absence or presence of dexamethasone. Concerning the molecular mechanism mediating the dexamethasone‐suppression of the TRAIL activity in pre‐osteoclasts, but not in leukemic cells, we found that dexamethasone induced a significant down‐regulation of the surface levels of TRAIL‐R2 in cells of the osteoclastic lineage but not in leukemic cells. The ability of dexamethasone to counteract the TRAIL pathway envisions a novel mechanism mediating the pro‐osteoclastic activity of dexamethasone in vivo. J. Cell. Physiol. 222: 357–364, 2010. © 2009 Wiley‐Liss, Inc.     

Induction of apogamy inEquisetum arvense

InEquisetum arvense, apogamous sporophytes were produced on medium containing 5×10^?6–5×10^?8 g/ml kinetin. NAA, IAA, GA₃, glucose and saccharose were ineffective for the induction of apogamy. On medium containing 5×10^?7–5×10^?8 g/ml kinetin, the gametophytes passed into sporophytic structures directly. On medium containing 5×10^?6 g/ml kinetin, some gametophytes passed into sporophytic structures directly, and others became a callus-like cell mass from which an apogamoun shoot arose. The results of the morphological observations on them were reported and compared with the sexually produced sporophyes. The apogamous sporophytes induced by 5×10^?7 g/ml kinetin were haploid in their nuclear phase and some of those induced by 5×10^?6 g/ml kinetin had a tendency to become diploid.     

Induction of sister chromatid exchanges in Chinese hamster cells by carcinogenic mutagens requiring metabolic activation

The induction of SCEs has proven to be the most sensitive mammalian system for detecting the effects of mutagenic carcinogens. Several chemicals that are mutagenic in the exquisitely sensitive Salmonella mutagenesis test have now been tested in Chinese hamster ovary (CHO) cells in culture. Cells were grown for 24 h (two rounds of DNA replication) in the presence of bromodeoxyuridine (Brd Urd) to form harlequin chromosomes in which it is possible to see the SCEs. To test whether the chemicals increase SCEs without metabolic activation, they were added at various concentrations for the entire culture period. To test if they induce SCEs after activation they were added for 30 min along with microsomes from rat liver (S-9 Mix of Ames). After this treatment the cells were cultured with Brd Urd. N-hydrosy-2-acetylamino-fluorene (10^?6?10^?4 M), N-acetoxy-2-acetylaminofluorenee (10)^?9?10^?7 M), and aflatoxin B₁ (10^?6?10^?4 M) all increased the yield of SCEs with increasing concentration. Further, aflatoxin B₁ was dramatically activated by the addition of rat liver microsomes. Benzo(a)pyrene (10^?6?10^?4 M), however, gave an increase only when activated. 2-aminofluorene (10^?6?10^?4 M) gave a slight increase only after long treatments without activation. In no case did 2-acetylamino-fluorene (10^?6?10^?4 M) increase SCE's. It thus appears that some of the chemicals that are positive in the Salmonella system are negative in the mammalian SCE system. Whether this reflects a difference in sensitivity between the two tests or the ability of the SCE test to discriminate between those chemicals that are active in bacteria, but not in mammals, is as yet unknown.     

Hydrocortisone stimulates fibronectin synthesis in cultured fibroblasts

The effect of hydrocortisone on fibronectin synthesis was investigated in cultured skin fibroblasis. Confluent cells were treated with hydrocortisone (10^?7 M to 10^?5 M) for 2 days and labeled with [³H]proline for 24 h. Fibronectin levels in both the culture medium and the cell layer were studied by gelatin-Sepharose affinity chromatography and SDS-polyacrylamide gel electrophoresis. In control cultures of human fetal skin fibroblasts, fibronectin constituted 8% of the total labeled proteins in the medium. The proportion of fibronectin increased to 13.1% at 10^?7 M hydrocortisone, 15.5% at 10^?6 M and to 19.4% at 10^?5 M. The proportion of fibronectin associated with the cell layer remained at 2-3% of total [³H]prolne-labeled proteins and did not increase with hydrocortisone exposure. The stimulating effect of hydrocortisone on medium fibronectin was also demonstrated in cultured human newborn foreskin fibroblasts and in rabbit skin fibroblasts.     

The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway Spruce)

Embryos of Picea abies at various developmental stages were cultured on defined media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (10^?5 M) and N⁶-benzyladenine (BA) (5×10^?6 M). The immature embryos gave rise to a highly friable and embryogenic callus which could be maintained by subculture and contained polarized and organized structures (somatic embryos) consisting of long highly vacuolated cells at one end (suspensor) and a group of small meristematic cells at the other (embryonal end). These structures closely resembled the early stages of normal zygotic embryogeny. Upon further culture these structures formed a bipolar shoot-root axis with an independent and closed vascular system. In many instances either the shoot or the root meristems failed to differentiate. Embryogenic tissues obtained on agar media could be transferred to liquid media and maintained by subculture for at least 6 months. The development of somatic embryos was observed in the liquid cultures also.     

Effect of salicylic acid on glucucorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma

The binding activity of [³H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [³H]dexamethasone of 3.64 · 10^?8 M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 · 10⁵.Total binding activity to [³H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid at 37°C. The maximum enhancement was seen at the concentration of 10^?3 M and in 3 h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicylic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin, also enhanced the total binding activity to [³H]dexamethasone of the cytoplasmic fraction at the concentration of 2 · 10^?5 M and 2 · 10^?7 M, respectively.