Increased capsaicin content in PFP-resistant cells of chili pepper (Capsicum annuum L.)

Cell suspensions of chili pepper (Capsicum annuum L.) were subjected to a selection process on semisolid medium containing the amino acid analog p-fluorophenylalanine (PFP). Four cell lines with different degrees of resistance were selected and suspension cultures were established from each of them. Resistance was retained even after 75 days of culture in the absence of PFP. PFP-resistant cell lines accumu lated higher levels of capsaicin than sensitive lines even after prolonged culture in PFP-free medium. Capsaicin production in non-selected cells was only 26.8% of that found in one cell line resistant to 500 M PFP. The capsaicin content in the non-selected cell suspension and in one of the resis tant cell lines was 6.7% and 24.9% respectively, that of fruits.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - PFP p-fluorophenylalanine - d. wt. dry weight - f. wt. fresh weight     

Phenylalanine ammonia-lyase activity and capsaicin-precursor compounds in p-fluorophenylalanine-resistant and -sensitive variant cells of chili pepper (Capsicum annuum)

Cell suspension cultures of chili pepper ( Capsicum annuum L. cv. Tampiqueño 74) displaying differences in their resistance to p -fluorophenylalanine (PFP) and in their contents of capsaicin (the compound which is responsible for the hot taste of chili pepper fruits) were characterized in relation to the activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the levels of free l -phenylalanine, phenolics and the phenylpropanoid acids involved in capsaicin biosynthesis. A nonselected cell line, a sensitive line (CA-02), a moderately resistant cell line (CA-29) and two resistant cell lines (CA-04 and CA-16) were studied. Higher PAL activities and higher levels of phenylalanine and phenolics were found in the PFP-resistant cells even after a minimum of 9 subcultures (15 days each) in the absence of the analog, indicating that the selected trait was stable. PFP-resistant chili pepper cells accumulated higher amounts of capsaicin precursors (cinnamic, caffeic and ferulic acids) than either the nonselected cells or the sensitive cell line. p -Coumaric acid was not detected at significant levels in any of the cell cultures. Overall, accumulation of free phenyl-alanine correlated well with PAL activity, phenolics, phenylpropanoids and capsaicin levels, suggesting an active flow through the phenylpropanoid pathway in PFP-resistant cells of chili pepper.     

Metabolism of p-fluorophenylalanine in p-fluorophenylalanine sensitive and resistant tobacco cell cultures

The metabolism of D- and L-p-fluorophenylalanine (PFP) in DL-PFP resistant and sensitive tobacco cell cultures (Nicotiana tabacum), cell lines TX4 and TX1, respectively, has been compared. The amino acid analogue was taken up at a lower rate by the resistant cell line TX4. Incorporation of PFP into protein was also considerably reduced in TX4 cells, compared to TX1 cells. This, however, resulted mainly from a diminished availability of PFP due to a more rapid conversion of PFP by TX4 cells. TX1 cells and TX4 cells converted PFP qualitatively in the same way. The only detectable metabolite of D-PFP was N-malonyl-D-PFP, while all metabolites of L-PFP were identified as sequent products of the initial deamination of L-PFP by the enzyme phenylalanine ammonia-lyase (PAL). As TX4 cells were endowed with higher PAL-activity than TX1 cells, the resistant cells were able to metabolize L-PFP more rapidly to give, e.g., p-fluorocinnamoyl glucose ester and p-fluorocinnamoyl putrescine. In the presence of the specific PAL-inhibitor -aminooxy--phenylpropionic acid TX4 cells were slightly more sensitive to PFP. This suggests that the better detoxification contributes to the acquired resistance. The use of PFP as specific indicator for cell lines with increased PAL-activity, and hence increased levels of phenolic compounds, is discussed.Abbreviations AOPP -aminooxy--phenylpropionic acid - MCW methanol:chloroform:water - PAL phenylalanine ammonia-lyase - PFP p-fluorophenylalanine - Phe phenylalanine     

Neuroprotective Effects of Chalcones from <Emphasis Type="Italic">Myracrodruon urundeuva</Emphasis> on 6-Hydroxydopamine-Induced Cytotoxicity in Rat Mesencephalic Cells

In the present work, we showed that a chalcone-enriched fraction (CEF) isolated from the stem bark of a Brazilian medicinal plant, Myracrodruon urundeuva, presents neuroprotective actions on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death, in rat mesencephalic cells. In the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] assay, which is an index of cell viability, CEF (1–100 μg/ml) reversed in a concentration-dependent manner the 6-OHDA-induced cell death. While cells exposed to 6-OHDA (40 μM) showed an increased concentration of thiobarbituric acid reactive substances (TBARS), the pretreatment with CEF (10–100 μg/ml) significantly decreased the 6-OHDA-induced TBARS formation, indicative of a neuroprotection against lipoperoxidation. Furthermore, the drastic increase of nitrite levels induced by 6-OHDA, indicative of nitric oxide formation and free radicals production, was prevented by CEF. Double staining with acridine orange/ethidium bromide showed that cultures exposed to 6-OHDA (40 and 200 μM) presented an increase of apoptotic and necrotic cell numbers in a concentration-dependent manner. CEF (100 μg/ml) protected cells from apoptosis and necrosis and increased number of cells presenting a normal morphology. The immunohistochemical analysis for tyrosine hydroxylase (TH) positive neurons indicated that 6-OHDA (40 and 200 μM) caused a concentration-dependent loss of TH+ and TH− neurons. CEF protected both cells types from 6-OHDA-induced cell death. All together, our results demonstrated neuroprotective effects of chalcones, which are able to reduce oxidative stress and apoptotic injury caused by 6-OHDA. Our findings suggest that chalcones could provide benefits, along with other therapies, in neurodegenerative injuries, such as Parkinson’s disease.     

The effect of oxidative stress on the production of the recombinant protein, interferon γ, produced by Chinese hamster ovary cells in stirred-batch culture

The CHO320 cell line, engineered to produce human interferon γ was investigated with regard to its susceptibility to oxidative stress. Batch cultures of the cells grown in a bench-top bioreactor exhibited no marked response to changes in oxygen concentration between 6% and 14% whereas cell growth and recombinant protein production were inhibited by increasing the oxygen to 20%. High concentrations of hydrogen peroxide (in excess of 200 μM) were required to inhibit growth of the CHO320 cells whereas concentrations of 50 μm and 100 μM had no effect on recombinant protein production. Buthionine sulphoximine (50 μM and 100 μM) completely depleted the cells of glutathione within 24 h; however, no quantitative effect on recombinant protein production was seen. It is concluded that the CHO320 cells are, possibly as a consequence of the long selection process they have undergone, very resistant to oxidative stress. Received: 14 November 1996 / Received last revision: 14 April 1997 / Accepted: 19 April 1997     

In vitro cold storage of cork oak shoot cultures

Friable callus cultures were initiated from cotyledons and hypocotyls of Opuntia ficus-indica. Explants from cotyledons produced significantly more callus than those from hypocotyls. Optimum callus growth was observed on Murashige & Skoog medium supplemented with 0.9 μM 6-furfurylaminopurine, 2.3 μM 2,4-dichlorophenoxyacetic acid, 1.0 μM 4-amino 3,5,6-trichloropicolinic acid, 400 mg l^-1 casein hydrolysate and 3% sucrose. The same medium without agar was used for establishing cell suspensions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Population responses in tobacco cell cultures during selection for resistance to para-fluorophenylalanine

Growth of cell suspension cultures of Nicotiana tabacum L. line TX1 is strongly inhibited by concentrations of para-fluorophenylalanine (PFP) >300 μM. Through step-wise selection, lines capable of growing in 450 μM PFP can readily be obtained. The manner in which the initial resistant variant population arises, however, is not consistent with a model involving rare appearance of individual mutant cells within the population. The latter model is also not supported by the results of reconstruction experiments which show that a very substantial subpopulation of resistant cells must be present in order to allow a growth response in the presence of the selection agent.     

Induction of apoptosis in prostate tumor PC-3 cells and inhibition of xenograft prostate tumor growth by the vanilloid capsaicin

Capsaicin, the pungent ingredient of hot chilli pepper, has been recently shown to induce apoptosis in several cell lines through a not well known mechanism. Here, we investigated the role of the vanilloid capsaicin in the death regulation of the human cancer androgen-resistant cell line PC-3. Capsaicin inhibited the growth of PC-3 with an IC₅₀ of 20 μM cells and induced cell apoptosis, as assessed by flow cytometry and nuclei staining with DAPI. Capsaicin induced apoptosis in prostate cells by a mechanism involving reactive oxygen species generation, dissipation of the mitochondrial inner transmembrane potential (ΔΨ_m) and activation of caspase 3. Capsaicin-induced apoptosis was not reduced by the antagonist capsazepine in a dose range from 0.1 μM to 20 μM, suggesting a receptor-independent mechanism. To study the in vivo effects of capsaicinoids, PC-3 cells were grown as xenografts in nude mice. Subcutaneous injection of either capsaicin or capsazepine (5 mg/kg body weight) in nude mice suppressed PC-3 tumor growth in all tumors investigated and induced apoptosis of tumor cells. Our data show a role for capsaicin against androgen-independent prostate cancer cells in vitro and in vivo and suggest that capsaicin is a promising anti-tumor agent in hormone-refractory prostate cancer, which shows resistance to many chemotherapeutic agents.     

Regeneration through organogenesis in rattan

Organogenic cultures were induced from zygotic embryo and megagametophyte explants of the Central American cycad species, Dioon edule. Plant growth medium consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l⁻¹ glutamine, 100 mg l⁻¹ arginine, 100 mg l⁻¹ asparagine, 60 g l⁻¹ sucrose, 8 g l⁻¹ Difco Bacto agar and was supplemented with kinetin (0 – 13.94 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0 – 9.05 μM) arranged as a 5×4 factorial in a randomized block design. Callus initiation occurred on a wide range of medium formulations from megagametophyte explants; however, shoot formation occurred only on medium supplemented with 2.26 μM 2,4-D. In comparison, callus initiation from explanted zygotic embryos occurred on fewer medium formulations, and adventitious shoot induction occurred from callus on formulations with 9.29–13.94 μM kinetin + 0.45–9.05 μM 2,4-D. Rooted shoots, derived from megagametophyte and zygotic embryo cultures, have been regenerated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Multiple shoot induction and callus regeneration in Sarcostemma brevistigma Wight & Arnott, a rare medicinal plant

An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA (0.5–8 μM) or Kn (0.5–8 μM) alone or in combination with NAA (0.5–1.5 μM). Maximum multiple shoot induction was observed on MS medium supplemented with 4 μM BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of 1 μM NAA along with 4 μM BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA (5 μM) and 2,4-D (2 μM). The callus was subcultured on MS medium supplemented with BA (2–15 μM) or Kn (2–15 μM) alone or in combination with NAA (0.5–2 μM) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA (1–7 μM) or IBA (1–7 μM). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.     

Comparative evaluation of antioxidant, nitrite scavenging, and antitumor effects of Antrodia camphorata extract

The effects of decoctions prepared from Antrodia camphorata on antioxidant, nitrite scavenging, and antitumor activities were investigated. Glutathione (GSH) production was 18.2 μM/g of rat liver under the influence of a methanol extract, a result similar to with the effect of silymarin administration. GSH peroxidase activity was about 2.0-fold higher than with silymarin administration. Superoxide dismutase activity was 18.9 U/mg protein, about 2.5-fold higher than the control group. The hot water extract (1000 μg/mL) showed the highest nitrite scavenging activity at 98.1%, similar to those observed with BHA and vitamin E. Among a variety of human cancer cell lines, when the concentration of hot water extract was increased from 31 to 500 μg/mL, the viability of Hep3B cell was decreased from 100 to 60.7%. On the other hand, in the case of methanol extract, it was decreased from 98.4 to 10.0%. These results supported the conclusion that the antioxidant, nitrite scavenging, and antitumor activities of this A. camphorata methanol extract indicate a potential source for the development of various health supplements and pharmaceutical and nutraceutical applications.     

Correlation between Phenylalanine Ammonia Lyase Activity and Phenolic Biosynthesis in p-Fluorophenylalanine-sensitive and -resistant Tobacco and Carrot Tissue Cultures

Phenylalanine ammonia lyase (PAL) activity was measured in p-fluorophenylalanine (PFP)-sensitive and -resistant tobacco (Nicotiana tabacum L.) and carrot (Daucus carota L.) cell lines which are known to oversynthesize phenylalanine. A correlation between phenolic levels and PAL activities was detected. The phenylalanine analog-resistant and -sensitive carrot cells showed no differences in the accumulation of phenolic compounds and PAL activities. The PFP-resistant tobacco cells, however, had 10 times higher levels of phenolics and also 10 to 20 times higher PAL activities than the PFP-sensitive line. The PAL activity in the resistant tobacco line increased dramatically after inoculation of the cells into fresh medium. Conditions affecting this increase were characterized.     

Selection of cell lines for the production of rosmarinic acid from cell suspension cultures of Orthosihon stamineus benth

Summary Cell suspension cultures of Orthosiphon stamineus were established from friable calluses produced from leaf pieces of in vitro plantlets that were derived from nodal segments of the mother plants collected from three different geographical locations. Eight lines were eventually selected after seven subculture cycles based on the growth characteristic (plant height) of the plantlets from the three locations: two fast-growing lines (>5.1 cm tall), three intermediate-growing lines (3.1–5.0 cm tall), and three slow-growing lines (<3.0 cm tall). All eight lines grew well in liquid Murashige and Skoog medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.4 μM 1-naphthaleneacetic acid (NAA). All cell lines exhibited the same growth pattern but produced different maximum cell biomass when cultured in this medium. The time of harvesting the plant cells from the culture medium and the geographical source of the original plant material were both found to affect the production of rosmarinic acid (RA) in cell cultures. Two cell lines were successfully selected and identified to produce high amounts of RA. These cell lines were a fast-growing cell line from Air Itam, Penang and an intermediate-growing cell line from Relau Agriculture Research Centre, Penang which could produce 5% [(w/w) dry weight] and 4.5% [(w/w) dry weight] of RA, respectively.     

MDMA (Ecstasy) Decreases the Number of Neurons and Stem Cells in Embryonic Cortical Cultures

Ecstasy, 3,4-methylenedioxymetamphetamine (MDMA), is a recreational drug used among adolescents, including young pregnant women. MDMA passes the placental barrier and may therefore influence fetal development. The aim was to investigate the direct effect of MDMA on cortical cells using dissociated CNS cortex of rat embryos, E17. The primary culture was exposed to a single dose of MDMA and collected 5 days later. MDMA caused a dramatic, dose-dependent (100 and 400 μM) decrease in nestin-positive stem cell density, as well as a significant reduction (400 μM) in NeuN-positive cells. By qPCR, MDMA (200 μM) caused a significant decrease in mRNA expression of the 5HT3 receptor, dopamine D₁ receptor, and glutamate transporter EAAT2-1, as well as an increase in mRNA levels of the NMDA NR1 receptor subunit and the 5HT_1A receptor. In conclusion, MDMA caused a marked reduction in stem cells and neurons in embryonic cortical primary cell cultures, which was accompanied by changes in mRNA expression of specific receptors and transporters for glutamatergic and monoaminergic neurotransmitters.     

Elimination of the yeastCandida parapsilosis from lymphoid cells and monolayer cells in culture

Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×10⁵ cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not detectable by light microscopy, can be removed by the addition of macrophages (2×10⁵/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost. This work was supported by Veterans Administration Research Funds.     

The use of human epidermal keratinocytes in culture as a model for studying the biochemical mechanisms of sulfur mustard toxicity

Human epidermal keratinocytes in culture were studied to evaluate their usefulness in demonstrating toxic events following exposure to sulfur mustard. Exposure of keratinocytes to sulfur mustard over a concentration range of 1–1000 μM HD, reduced NAD+ levels from 96% to 32% of control levels. When keratinocytes were exposed to a concentration of 300 μM HD, NAD+ levels began to fall at 1 hour and reached a plateau of 47% of control levels at 4 hours. Niacinamide, an inhibitor of the enzyme poly(ADP-ribose) polymerase, partially protected mustard-exposed cells against NAD+ depletion. It also protected cellular viability as assessed by vital staining 24 hours after exposure. This protection was not seen in long-term (72 hr) cultures. These studies suggest that human epidermal keratinocytes in culture can serve as a usefulin vitro model for research into the biochemical mechanisms of sulfur mustard-induced cutaneous injury.     

Modulating zucchini cotyledon plate meristem activity by interactions between the cycline-dependent kinase inhibitor roscovitine and cytokinins

The inhibitors of cytokinin N-glucosylation are known to influence the growth of some plant objects including cotyledons. The use of the plate meristem of zucchini cotyledon as an experimental system allowed us to study for the first time the way in which the changes in the cell division are integrated in this growth reaction. Roscovitine, a potent inhibitor of cytokinin N-glucosylation and cycline-dependent kinases, did not show to have an effect on the meristem activity when applied in 100 μM to cultivated zucchini cotyledons, and acted as an inhibitor in concentrations higher than 400 μM. A 200 μM roscovitine stimulated both palisade cell division and growth. In different seed batches, 400 μM roscovitine acted as a stimulator or an inhibitor. A much stronger stimulating effect on growth and cell division was observed after application of benzyladenine (BA, 10 μM). In contrast to BA, roscovitine provoked a formation of principally flat lamina. In combined treatments, it lowered the stimulating effect of BA; 400 μM roscovitine combined with BA severely suppressed the growth and division activity. This cellular behavior and changes in cotyledon growth could be due to the roscovitine-provoked changes in endogenous cytokinin levels via the inhibition of cytokinin N-glucosylation. Roscovitine-caused stimulation of cell growth and division is stronger in the marginal meristem than that registered in central regions of the cotyledon blade. In this region it also changed the pattern of cell division and lowered the adhesion between the clusters, which enhanced the appearance of local ruptures of the cotyledon edges. The first palisade layer of the plate meristem of cultured zucchini cotyledons, the natural mono-layer of proliferating palisade cells, may be used for screening the inhibitors of cycline-dependent kinases and cytokinin N-glucosylation with regard to their effects on cell division and growth.     

In vitro culture of Phyllanthus caroliniensis (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus caroliniensis (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication (21–23 shoots per explant) was achieved on MS or AR media supplemented with either 5.0 μM BA, 1.25–5.0 μM kinetin or 2.5–5.0 μM 2iP. Rooting was achieved with 80–100% of the microshoots on MS medium without growth regulators, although 1.25 μM NAA and 1.25–5.0 μM IAA promoted significant increases in the number of roots per explant. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed on in vitro grown plantlets and after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated in the vertical position on MS medium supplemented with 5.0 μM 2,4-D. Root cultures were successfully established on MS medium containing 1.1 μM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Relative resistance to methothexate by proliferating normal rabbit epidermal cells in vitro

Summary Primary cell cultures of normal rabbit epidermal cells (keratinocytes) were established without the use of enzymatic techniques. Six experiments were carried out on cells from six different rabbits. When these cells were exposed to methotrexate (MTX) for 24 h at 1 μg/ml, proliferation, as measured by cells entering mitosis, was significantly inhibited (P<0.05) in only one experiment. When the dose of MTX was elevated to 100 μg/ml, only two experiments showed significant inhibition of mitosis. This minimal inhibition of mitosis by MTX was contrasted by the dramatic inhibitory effect of this antimetabolite on DNA synthesis. At 1 μg/ml MTX for 24 h, DNA synthesis, as measured by [³H]deoxyuridine uptake, was inhibited >95%. We can conclude that under certain conditions, the rabbit keratinocyte may represent a normal cell type that is inherently resistant to the antiproliferative effects of methotrexate. The research was supported by National Cancer Institute Grant CA 11536.