Selection of Variants with High Levels of Biotin from Cultured Green Lavandula vera Cells Irradiated with Gamma Rays

Cultured green Lavandula vera cells were irradiated with variousdosages of gamma rays which increased the variation in the amountof free biotin produced by the cell clones. Variant sublinescontaining much more free biotin than the original line wereobtained by repeated selection. The effectiveness of gamma raysfor the induction of the variant sublines is described. (Received June 2, 1982; Accepted September 14, 1982)     

Characterization of Carrot and Tobacco Cell Cultures Resistant to p-Fluorophenylalanine

This study describes the isolation and characterization of p-fluorophenylalanine-resistant diploid tobacco (Nicotiana tabacum L.) and diploid carrot (Daucus carota L.) cultured cell lines. The p-fluorophenylalanine-resistant tobacco and carrot lines can grow in medium containing p-fluorophenylalanine concentrations 10 to more than 100 times those which inhibit the growth of susceptible cells, respectively. The resistance trait was retained when the cells were grown in a medium lacking the phenylalanine analog for 50 generations. All 14 single cell clones started from the resistant carrot line remained resistant. The resistant lines incorporated much less p-fluorophenylalanine into protein, partially due to a decrease in uptake. In carrots, an increase in the levels of free phenylalanine and tyrosine also apparently contributed to the decreased incorporation of p-fluorophenylalanine into protein by increasing the metabolic pool size which diluted the incoming analog and caused a lowered percentage of incorporation, which was observed. Apparently, phenylalanine and tyrosine synthesis was also increased in resistant tobacco lines, since chorismate mutase was found to have greater activity and to be less sensitive to inhibition by phenylalanine, tyrosine, and p-fluorophenylalanine. It appears, however, that phenylalanine and tyrosine do not accumulate above the normal levels in the resistant tobacco cells, as these amino acids were apparently converted into phenolic compounds which were found in higher levels (6 times). The low frequency of appearance, the stability of the trait, and the biochemical nature of the resistance, indicate that the p-fluorophenylalanine resistance found in the carrot and tobacco lines described here is due to a mutation.     

Metabolic and structural changes during early maturation of Inga vera seeds are consistent with the lack of a desiccation phase

Inga vera, native to South America, is an important leguminous species used for ecological restoration of riparian forests and its seeds are among the most recalcitrant ones described up to date. In this work, we analysed the metabolic profile, cell ultrastructure as well as cell wall polysaccharides of I. vera seeds in order to better understand its maturation, which allows embryo germination without a quiescent phase. Increased amounts of citric, glutamic, pyroglutamic, and aspartic acids from stages I to II (120 and 129 days after flowering (DAF)) corroborate the hypothesis of high metabolism, shifting from fermentative to aerobic respiration at seed maturity. This phase was characterized by an extensive vacuolization of embryonic cells, which also indicate high metabolic activity. The proportion of arabinose in the cell walls of embryonic axis (approx. 20%) was lower than those found in some orthodox seeds (nearly 40%), suggesting that arabinose-containing polysaccharides, which are thought to provide more flexibility to the cell wall during natural drying, are less abundant in I. vera seeds. Taken together, our results provide evidence that the major changes occurred during early stages of seed maturation of I. vera, indicating that the rapid temporary metabolic shift observed between stages I and II may be related to the lack of desiccation phase, moving directly to germination.     

Induction, selection and isolation of auxin heterotrophic and auxin-resistant mutants from cultured crown gall cells irradiated with gamma rays

Cultured crown gall cells were irradiated with gamma rays toinduce mutation in indoleacetic acid biosynthesis. The irradiatedcells were plated on a selection medium which contained auxin.Mutant cells adapted to selection media were characterized asauxin-heterotrophic and auxin-resistant cell lines. The auxin-heterotrophicmutants contained little auxin, whereas the auxin-resistantand -autotrophic mutants contained large amounts of auxin evenwhen cultured with 0.3 ppm of 2,4-dichlorophenoxyacetic acid.Each mutant cell line contained as much octopine as its parentalcells. The mutation rate was calculated as in the order of 10^–8. (Received May 6, 1980; )     

Expression of 5-Methyltryptophan Resistance in Plants Regenerated from Resistant Cell Lines of Datura innoxia

Seventy-nine 5-methyltryptophan-resistant cell lines have been selected from haploid Datura innoxia Mill. cell cultures by plating suspensions in agar medium containing a growth inhibitory concentration of 5-methyltryptophan. Mutagen treatment increased the frequency of resistance. The eleven variants tested posses an altered anthranilate synthase less sensitive to feedback inhibition by tryptophan. All five of the variants which were analyzed for free amino acids contained elevated levels of free tryptophan (8 to 30 times the wild type level). None of the selected cell lines were auxin-autotrophic. Resistance to 5-methyltryptophan, altered anthranilate synthase, and high free tryptophan (4 to 44 times) were also expressed in leaves of plants regenerated from the variant lines and in cultures reinitiated from the resistant plants. These results show that the amino acid overproduction phenotype can be selected at the cellular level of organization and be expressed identically in whole plants regenerated from the selected cells.     

Preparation of Polyamide Nanocapsules of Aloe vera L. Delivery with In Vivo Studies

Aloe vera is the oldest medicinal plant ever known and the most applied medicinal plant worldwide. The purpose of this study was to prepare polyamide nanocapsules containing A. vera L. by an emulsion diffusion technique with in vivo studies. Diethyletriamine (DETA) was used as the encapsulating polymer with acetone ethyl acetate and dimethyl sulfoxide (DMSO) as the organic solvents and Tween and gelatin in water as the stabilizers. Sebacoyl chloride (SC) monomer, A. vera L. extract, and olive oil were mixed with the acetone and then water containing DETA monomer was added to the solution using a magnetic stirrer. Finally, the acetone was removed under vacuum, and nanocapsules were obtained using a freeze drier. This study showed that the size of the nanocapsule depends on a variety of factors such as the ratio of polymer to oil, the concentration of polymers, and the plant extract. The first sample is without surfactant and the size of nanocapsules in the sample is 115 nm. By adding surfactant, nanocapsules size was reduced to 96 nm. Nanocapsules containing A. vera were administered to rats and the effects were compared with a normal control group. The results showed that in the A. vera group, the effect is higher. The nanocapsules were identified by scanning electron microscopy (SEM), zeta potential sizer (ZPS), and Fourier-transform infrared spectroscopy (FT-IR).KEY WORDS: Aloe vera L., in vivo, medicinal plant, nanocapsule, polyamide nanocapsule     

Characterization of cultured tobacco cell lines resistant to ethionine, a methionine analog

Two cultured tobacco cell lines (Nicotiana tabacum L. cv Xanthi) were selected for resistance to growth inhibition by the methionine analog ethionine. Comparison of the free amino acid pool levels in these lines with those of the ethionine-sensitive parental line showed substantial accumulation of methionine (110×), threonine (18×), and lysine (5×). In vitro enzymic analysis of lysine-sensitive aspartate kinase activity showed the resistant lines to contain 16 times that found in the sensitive line. The lysine-sensitive enzymes from both resistant and sensitive lines coeluted from DEAE-cellulose and exhibited similar K_m values. Both showed identical lysine plus S-adenosylmethionine inhibition profiles suggesting that the elevated activity in the resistant lines is not due to a structural change in the lysine-sensitive enzyme but possibly to the level of its expression.     

Physiology of Hormone Autonomous Tissue Lines Derived From Radiation-Induced Tumors of Arabidopsis thaliana

γ-Radiation-induced tumors of Arabidopsis thaliana L. have been produced as a novel approach to isolation of genes that regulate plant development. Tumors excised from irradiated plants are hormone autonomous in culture and have been maintained on hormone-free medium for up to 4 years. Five tumor tissue lines having different morphologies and growth rates were analyzed for auxin, cytokinin, and 1-aminocyclopropane-1-carboxylic acid (ACC) content, ethylene production, and response to exogenous growth regulators. Normal tissues and two crown gall tissue lines were analyzed for comparison. Rosettes and whole seedlings each contained approximately 30 nanograms· (gram fresh weight)⁻¹ free indoleacetic acid (IAA), 150 nanograms· (gram fresh weight)⁻¹ ester-conjugated IAA, and 10 to 20 micrograms· (gram fresh weight)⁻¹ amide-conjugated IAA. The crown gall lines contained similar amounts of free and ester-conjugated IAA but less amide conjugates. Whereas three of the radiation-induced tumor lines had IAA profiles similar to normal tissues, one line had 10- to 100-fold more free IAA and three- to 10-fold less amide-conjugated IAA. The fifth line had normal free IAA levels but more conjugated IAA than control tissues. Whole seedlings contained approximately 2 nanograms· (gram fresh weight)⁻¹ of both zeatin riboside and isopentenyladenosine. The crown gall lines had 100- to 1000-fold higher levels of each cytokinin. In contrast, the three radiation-induced tumor lines analyzed contained cytokinin levels similar to the control tissue. The radiation-induced tumor tissues produced very little ethylene, although each contained relatively high levels of ACC. Normal callus contained similar amounts of ACC but produced several times more ethylene than the radiation-induced tumor lines. Each of the radiation-induced tumor tissues displayed a unique set of responses to exogenously supplied growth regulators. Only one tumor line showed the same response as normal callus to both auxin and cytokinin feeding. In some cases, one or more tumor lines showed increased sensitivity to certain growth substances. In other cases, growth regulator feeding had no significant effect on tumor tissue growth. Morphology of the radiation-induced tumor tissues generally did not correlate with auxin to cytokinin ratio in the expected manner. The results suggest that a different primary genetic event led to the formation of each tumor and that growth and differentiation in the tumor tissue lines are uncoupled from the normal hormonal controls.     

Phosphatidylcholine Synthesis in Castor Bean Endosperm : I. Metabolism of l-Serine

Three levels of free amines and the activities of their biosynthetic enzymes were measured in subcellular fractions of two cell lines of Nicotiana tabacum L. cv Xanthi. The TX4 cell line, a p-fluorophenylalanine resistant culture which accumulates high levels of cinnamoylamides, was compared to the wild-type culture TX1. In cells harvested on day 6 of the growth cycle, nearly all free putrescine, spermidine, and tyramine was found in the supernatant fraction of both cell lines. Although a consistent portion of ornithine decarboxylase activity was detected in the nuclear-enriched fractions of TX1 and TX4, the largest levels of activity were in the supernatants of both lines. In TX1, arginine decarboxylase activity was low relative to that of ornithine decarboxylase, but, in the TX4 line arginine decarboxylase levels in the cytosol were substantially elevated. Tyrosine decarboxylase was not detected in 6-day-old TX1 cells, but significant amounts of activity were measured in the 1000g and supernatant fractions of TX4. S-Adenosylmethionine decarboxylase activity was low in both cell lines and was located predominantly in the supernatant.     

Magnetic resonance imaging visualization of targeted cells by the internalization of supramolecular adducts formed between avidin and biotinylated Gd3+ chelates

The high binding affinity between avidin and biotin has been exploited to develop a procedure for magnetic resonance imaging (MRI) visualization of target cells. SHIN3 and PANC1 tumor cell lines have been used as target cells because they possess on their membranes galactosyl receptors able to bind avidin molecules. Avidin–Gd chelate adducts have been built by using two Gd complexes containing one (Gd-I) and two (Gd-II) biotin residues, respectively. The relaxivities of such supramolecular adducts are significantly higher than those shown by free Gd-I and Gd-II. There is evidence of the occurrence of multilayered adducts in which the bis-biotinylated Gd³⁺ complex acts as a bridge between adjacent avidin molecules. MRI differentiation of labeled versus unlabeled cells has been attained when approximately 6×10⁸ Gd units were internalized in each cell. Furthermore, there is a marked decrease in the measured intracellular T₁ relaxivity as the number of internalized Gd complexes increases, probably owing to too short relaxation times of endosomic water protons with respect to their diffusion lifetime.     

Fatty acid composition of lipids from differentiated tissues and cell cultures of Euonymus europaeus

The fatty acid patterns of Euonymus europaeus callus cultures and cell suspension cultures were analysed at the beginning of stationary growth phase and compared with those from the respective differentiated tissues. The lipid and fatty acid patterns in cell cultures differed remarkably from those in the tissues of the mother plant. No glycerol triacetate was detected in the callus cultures derived from differentiated tissues whereas in seeds this lipid compound amounts to 29%. In addition to fatty acids normally occurring in differentiated tissues, lipids in cultured cells also contained short-chain (C₁₂–C₁₄) as well as very long-chain fatty acids (C₂₀–C₂₄). In tissue culture cells the major fatty acids were found to be saturated, whereas in the mother cells unsaturated fatty acids were predominant. Palmitic acid is the most abundant fatty acid in most of the cultures. Lauric, myristic and palmitic acid amount to 50% in lipids of cell suspension cultures.     

Obtaining and Study of Callus and Suspension Plant Cell Cultures of <Emphasis Type="Italic">Tribulus terrestris</Emphasis> L., a Producer of Steroidal Glycosides

Callus and suspension plant cell cultures of Tribulus terrestris L., a valuable medicinal plant producing steroidal glycosides, were obtained. The seeds from an American population of T. terrestris were used as explants. Regulation of the production and growth of cell cultures, as well as the biosynthetic characteristics of the cell lines, were studied. The combination of phytohormones of 2,4-D (2.0 mg/L) and BAP (1.0 mg/L) was found to be optimal for callus induction and cultivation. Suspension cell culture obtained in liquid medium of the same composition showed such high growth characteristics during prolonged cultivation (more than 2 years) as a maximum accumulation of dry biomass of 13 g/L, specific growth rate at exponential phase of 0.24 day^–1, and economical coefficient of 0.39. A semicontinuous mode of cultivation was used to grow the plant cell suspension in a lab-scale bioreactor. Screening of the steroidal glycosides in the obtained cell cultures was carried out. Steroidal glycosides were not found in the callus cultures. However, as was demonstrated by TLC and UPLC ESI MS methods, the suspension culture contained furostanol glycosides, and their amount increased during the cultivation process. These results support the hypothesis of the autoselection of cultivated cells containing compounds promoting their proliferation in vitro.     

The effect of post-treatment with caffeine on survival and UV-induced mutation frequencies in Chinese hamster and mouse lymphoma cells in vitro

The effect of post-treatment with caffeine on the survival of a number of cell lines after UV-irradiation has been studied. The mouse lymphoma cell lines P388 and L5178YS were sensitized by caffeine but only after UV doses of 50 erg/mm² and above. V79 cells also showed sensitization by caffeine but CHO cells and two cell lines YS and YR derived from Yoshida sarcoma of rats, sensitive and resistant to UV radiation, respectively, showed no effect.P388 and V79 cells were both mutable by UV, and caffeine, when studied at a single expression time (42–48 h) and at a single dose level (0.5 M and 0.75 M, respectively) suppressed the UV-induced mutation frequency in both cell lines. L51788YS cells although sensitized by caffeine showed no increase in frequency of thymidine-resistant (TdR^r) colonies when irradiated with UV.On more detaled examination, caffeine was found to delay the expression of UV-induced mutations inV79 cells, and the delay was dependent on the dose of caffine used. The effect on expression time was less when caffeine was present 0–48 h than when it was present throughout the post-irradiation incubation period. Similar results were obtained in P388 cells.The data are discussed in relation to those of other workers and to the concept that caffeine inhibits an error prone post-replication repair process in mammalian cells     

Effects of aloe extracts on human normal and tumor cells in vitro

Fractions of leaf extracts from 2 local types, labeledAloe vera (subsequently identified asAloe barbadensis Mill, andA. saponaria Haw.), were prepared by differential centrifugation and tested by in vitro assays for the presence of lectinlike activities and for effects on the attachment and growth of human normal and tumor cells. Fractions of extracts of fresh leaves and commercially “stabilized”Aloe vera gel had high levels of lectin-like substances measured by immunodiffusion and hemagglutination assays. Substances in fluid fractions from both fresh leaf sources were found to markedly promote attachment and growth of human normal, but not tumor, cells and to enhance healing of wounded cell monolayers. In contrast, fractions of “stabilized”Aloe vera gel were equally cytotoxic for human normal and tumor cells in vitro. Results from cell assays suggested that the observed growth promotion and wound healing effects of aloe substances in vitro may be analogous to what has been observed in vivo during healing of wounds and burns.     

Microarray Analysis of Siberian Ginseng Cyclic Somatic Embryogenesis Culture Systems Provides Insight into Molecular Mechanisms of Embryogenic Cell Cluster Generation

Four systems of cyclic somatic embryogenesis of Siberian ginseng (Eleutherococcus senticosus Maxim) were used to study the mechanism of embryonic cell cluster generation. The first, direct somatic embryo induction (DSEI), generates secondary embryos directly from the primary somatic embryos; the second, direct embryogenic cell cluster induction (DEC)), induces embryogenic cell clusters directly from somatic embryos in agar medium. Subsequently, we found that when DEC-derived somatic embryos are transferred to suspension culture or a bioreactor culture, only somatic embryos are induced, and embryogenic cell clusters cannot form. Therefore, these new lines were named DEC cultured by liquid medium (ECS) and DEC cultured by bioreactor (ECB), respectively. Transmission electron microscopy showed that DEC epidermal cells contained a variety of inclusions, distinct from other lines. A cDNA library of DEC was constructed, and 1,948 gene clusters were obtained and used as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. EsXTH1 and EsPLT1, which were among the genes upregulated in DEC, were cloned using the rapid amplification of cDNA ends (RACE). Real-time quantitative PCR showed EsXTH1 was more highly expressed in DEC than in other lines throughout the culture cycle, and EsPLT1 expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that EsXTH1 and EsPLT1 may be the essential genes that play important roles during the induction of embryogenic cell clusters.     

Identification of avidin and streptavidin binding motifs among peptides selected from a synthetic peptide library consisting solely of D-amino acids

Peptides consisting solely of D -amino acids (D -peptides) as opposed to their L -counterparts (L -peptides) are resistant towards proteolytic degradation in the organism and may therefore be useful in future efforts to develop new stable peptide-based drugs. Using the random synthetic peptide library technique several L - and D -peptides, capable of binding to both avidin and streptavidin, were found. The L -peptides contained the previously described HPQ/M motifis, and among the D -peptides three binding motifs could be identified, of which the most frequently found one contained an N-terminal aliphatic hydrophobic amino acid (V, L or I) and an aromatic amino acid (Y or F) on the second position. At the third position in this motif several different amino acid residues were found, although N was the most frequent. Peptides representing two of the D -motifs were synthesized as well as peptides containing the HPQ/M motifs, and their binding properties were examined. Although the D -peptides were originally selected using avidin they also inhibited binding between immobilized biotin and soluble streptavidin as well as avidin. The IC₅₀ of some of the peptides were approximately 10⁵ times higher than the IC₅₀ for biotin but some had a lower IC₅₀ than iminobiotin. The D -peptides, which were originally selected from the library using avidin, could also inhibit the binding between streptavidin and biotin. Likewise, L -peptides selected from a library screened with streptavidin, could inhibit the binding of both streptavidin and avidin to immobilized biotin. Furthermore, the D -peptide, VFSVQSGS, as well as biotin could inhibit binding of streptavidin to an immobilized L -peptide (RYHPQSGS). This indicates that the biotin-like structure mimicked by these two seemingly very different peptides may react with the same binding sites in the streptavidin molecule.     

Relationship between cell surface protease activity and doubling time in various normal and transformed cells

A sensitive method for measuring cell surface and secreted protease activity utilizing ³H-labelled casein is described. The method is based upon proteolytic degradation of the casein substrate into trichloroacetic acid soluble ³H-labelled peptides. Utilizing the radioassay we found that all cultured cell lines examined contain cell surface proteolytic activity which is not secreted into the media. The protease activity was found to be due to protease(s) other than plasminogen activator or plasmin. A comparison of surface protease activity of normal and transformed mouse epidermal cells indicated that the transformed cells contained approximately 3–4 times more proteolytic activity than the normal cells.Surface protease activity was also correlated with the doubling times of various cultured cells. The results indicated that cultured cells with doubling times of greater than three days possess less surface protease activity than cells with shorter doubling times. In order to determine changes in the levels of surface protease activity during the cell cycle several cell lines were synchronized. In synchronized rabbit aortic fibroblasts, mouse transformed epidermal cells and human melanoma cells, a marked increase in surface protease activity was observed during or before mitosis. The protease levels decreased following mitosis. The results suggest that in culture, cell surface protease(s) may be important factor in regulating the rate of cell growth.     

A method for high-frequency intergeneric fusion of plant protoplasts

Summary Protoplasts of Vicia hajastana Grossh. obtained from suspension-culture cells and Pisum sativum L. obtained from leaves adhered tightly to each other in concentrated solutions of high-molecular-weight polyethylene glycol (PEG). The adhesion occurred non-specifically between the free protoplasts from the same species as well as from the different species and genus. It was enhanced by enrichment of the PEG solution with calcium. Very few heteroplasmic fusions occurred during the period when the protoplasts were incubated in the PEG solution. However, many heterokaryons (up to 10%) were formed soon after the PEG solution was diluted out. The same phenomena were also observed in protoplasts from suspension-culture cells of Glycine max L. and from leaves of Hordeum vulgare L. Vicia and soybean protoplasts obtained from cultured cells regenerated cell walls and underwent sustained cell division after such treatment. Some Vicia-pea heterokaryons divided once. Over 10% of the soybean-barley hybrids divided in 7 days. Some divided 4–5 times and formed small clusters of cells in 10 days. The hybrids were recognizable because they contained chloroplasts from the leaf protoplast and exhibited morphological characters typical of the chlorophyll-less cells. None of the protoplasts from pea and barley leaves, either with or without PEG treatment, underwent cell division during the period of observation. The mechanism of adhesion and fusion of the protoplasts has been discussed.National Research Council (Canada) No. 13732.     

Characterization of parameters influencing receptor-mediated endocytosis in cultured soybean cells

In a recent publication, we were able to demonstrate that biotin enters plant cells by receptor-mediated endocytosis and that impermeable macromolecules can be cotransported into cells by the same pathway if they are first covalently linked to biotin. In the present study, we have exploited the biotin endocytosis pathway to evaluate the variables in the cell wall and surrounding growth medium that influence the efficiency of endocytosis in plants. Under normal growth conditions, the major constraint limiting macromolecule endocytosis was found to be the size of the internalized macromolecule. Thus, a log-linear relationship with a negative slope exists between the molecular weight of the biotin-conjugated macromolecule and its rate of internalization by cultured soybean cells. This relationship, which extends from insulin (M_r approximately 5700) to immunoglobulin G (M_r approximately 160,000), is characterized by a slope of −1.04 × 10⁵ molecules/cell/min per log M_r unit and an x intercept (no endocytosis detectable) of approximately log 160,000 daltons. Unfortunately, mild digestion with cell wall-degrading enzymes is unable to increase significantly the upper size limit of molecules that can be internalized, but uptake of lower molecular weight proteins can be enhanced by mild cell wall digestion. The optimal extracellular pH for endocytosis was found to be 4.6, i.e. near the normal pH of the cell culture medium. Furthermore, the osmotic strength at which endocytosis occurs most rapidly was observed to be isotonic to slightly hypotonic, suggesting that turgor pressure within the plant cell must not be a major determinant of endocytosis rates by cultured soybean (Glycine max) cells. Finally, cell age was found to impact significantly on the rate of macromolecule internalization, with maximal uptake rates occurring during early exponential growth and decreasing by a factor of 2 when the cells reach stationary growth phase.     

Entrapment of Lavandula vera cells and production of pigments by entrapped cells

Finely suspended cells of Lavandula vera were obtained by cultivating the cells in the presence of 50 mM CaCl₂ and entrapped homogeneously in gels formed from natural polysaccharides, agar, alginate and κ-carrageenan. The gel-entrapped cells grew well inside the gel matrices, the growth being confirmed by the increases in oxygen uptake, cell number and chlorophyll content. Blue pigments were synthesized de novo in the presence of l-cysteine by the gel-entrapped cells as well as by the free counterparts. Calcium alginate gel-entrapped cells were employed for the repeated production of the pigments for over 7 months by alternating growth and production phases. Entrapment with adequate gels stabilized markedly the viability and the pigment productivity of the plant cells.