Isoenzyme changes during the growth cycle of paul's scarlet rose cell suspensions

A preliminary study has been made of the development of malate dehydrogenase, glutamate dehydrogenase, esterase and peroxidase activities during the growth cycle of Paul's Scarlet rose cells in batch propagated suspension cultures. These cells, which show no tendency to differentiate in culture, show quantitative changes in isoenzymes during the growth cycle. Qualitative changes were also observed, particularly in the case of peroxidase.     

Volatile components in cell suspension cultures of Cryptomeria japonica

The cell suspension culture of Cryptomeria japonica contains volatile oils, the yield of which was 0.005–0.01% of the fresh cells. In the volatiles, five aldehydes, ten fatty acids and their esters, and two diterpenes of abietatriene and ferruginol have been found. Of these, palmitic acid is present as the most predominant component, amounting to ca 40% of the volatiles.     

A microcomupter program for the computation of free energy of the secondary structure of RNA oligomers by Ninio's rules

An interactive microcomputer program for the rapid computation of the free (ΔG) of the secondary structure of RNA molecules is presented. The program assigns free energies (in kcal/mol) to helices; bulge, internal, and multi-branch loops; hairpins; unparied, and G:C termini according to Ninio's rules, and displays a running total during computation. It is written in ‘Microsoft Basic’ and is apllicable to virtually any Basic system with no modification. The program enables rapid bench-top determinations of free energies attributable to secondary structure features of RNA oligomers, and eliminates both the tedium and risk of error associated with manual calculations.     

Effect of norleucine on growth,protein and catechol oxidase synthesis in tobacco suspension cultures

Tobacco cells were grown in artificial media with defined amino acid composition. In such media, the addition of methionine or norleucine caused increases in the specific activity of the catechol oxidase, while in the normal medium norleucine depressed it. The differences of the effect of norleucine on synthesis of catechol oxidase and on cell growth is demonstrated, as is the reversibility of the norleucine effect by methionine. The incorporation of norleucine into a purified enzyme fraction is shown. The change in the electrophoretic patterns of the enzyme during growth in the absence and presence of norleucine was followed. [¹⁴C]-Leucine incorporation by control and norleucine treated cells was examined and it was shown that protein synthesis in the norleucine treated cells was markedly changed and total incorporation reduced. Incorporation into soluble protein was reduced, but increased in the 20 000 g precipitate fraction. Nevertheless use of autoradiography indicates that some catechol oxidase is apparently synthesised in the presence of norleucine.     

Regulatory effects of exogenous branched-chain amino acids in Nicotiana plumbaginifolia cell suspension cultures

Nicotiana plumbaginifolia suspension cultured cells were grown on medium supplemented with valine, leucine and isoleucine, singly or in combination. The effects of the three branched-chain amino acids on cell growth rate and on the activity of acetohydroxyacid synthase (AHAS), the first enzyme (and the main regulative site) of their biosynthetic pathway, were studied. Results showed that valine and leucine, at concentrations ranging from 10^–4 to 10^–3 M, inhibit growth, and at higher doses (from 10^–2 to 10^–1 M) AHAS activity. Growth, but not AHAS activity, was affected also by isoleucine. The addition of ammonium succinate to the culture medium, in order to counteract a possible general inhibitory effect of these compounds on nitrogen metabolism, relieved only partially their cytotoxicity. Feeding cells with equimolar mixtures of the three amino acids resulted in a minor but reproducible decrease in AHAS level, which was proportional to the dose. A similar result was obtained also on N. plumbaginifolia seedlings, suggesting that in this species a modulation of enzyme level could play a role in controlling the flow of metabolites through the pathway.Abbreviations AHAS acetohydroxyacid synthase - BCAA branched-chain amino acids - FAD flavin adenine dinucleotide - GS glutamine synthetase - TPP thiamine pyrophosphate     

Semicontinuous cultivation of photoautotrophic cell suspension cultures in a 20 l airlift-reactor

An airlift-bioreactor system was established for semicontinuous growth of photosynthetically active plant cell suspension cultures in a controlled environment. The bioreactor unit was constructed as a conventional, internal draught tube airlift-reactor, which is characterized by a H D^-1 ratio of 2.9, a ratio of the cross-sectional area of the riser to the cross-sectional area of the downcomer of 0.25 and a surface area of 0.435 m² for illumination. Cultivation experiments could be scaled up to working volumes of maximal 20 1. Sixteen fluorescent tubes were fixed around the outer glass cylinder to provide cells continuously with light. An external cooling device was used to keep the temperature constantly at 27°C. Agitation as well as supply with CO₂ was performed by injecting air enriched with CO₂ through a ring-shaped sparger at the bottom of the vessel. A first set of experiments was carried out with a photoautotrophic culture of Chenopodium rubrum L. Cell material adapted to large scale culture conditions was used to inoculate a modified MS medium (Murashige & Skoog 1962) without any organic constituents. Under these conditions a biomass increase of 1870% was achieved in 18 days. Several physiological parameters (e.g. pigmentation, photosynthetic O₂ evolution, carbohydrate content) were measured routinely to elucidate the growth characteristics of large-scale grown Chenopodium cells. Electron microscopic photographs from different phases of culture growth clearly demonstrate the pattern of cellular development. Special emphasis was placed upon the differentiation of chloroplast ultrastructure. The presented data confirm the feasibility of large-scale culture techniques with photosynthetic active plant cell cultures.Abbreviations D diameter - DW dry weight - FW fresh weight - H height - K_La volumetric oxygen transfer coefficient (h^-1) - MES 2-(N-morpholino)-ethanesulfonic acid - specific growth rate (d^-1) - PAR photosynthetically active radiation (400–700 nm) - Pepcase phosphoenolpyruvate carboxylase - Rubisco ribulose-1,5-biphosphate carboxylase/oxygenase - t_d doubling time (d) - vvm (aeration volume) (medium volume)^-1 min^-1 Dedicated to Prof. F.-C. Czygan on the Occasion of his 60th Birthday     

Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin

Cell growth of tumour ascites cell was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2–100 μg/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. Theses results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells whihc represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the ¹⁴C-labelled asialofetuin degradation. We can conclude that Ricinus lectic present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.     

Biosynthesis of morindone and alizarin in intact plants and cell suspension cultures of Morinda citrifolia

Chrysophanol (1,8-dihydroxy-3-methylanthraquinone) is an acetate-derived anthraquinone which is substituted in both benzenoid rings. Morindone (1,5,6-trihydroxy-2-methylanthraquinone) is also substituted in both rings but is shown to be derived from shikimic acid via o-succinoylbenzoic acid.     

On the role of S: Sulfotransferases in assimilatory sulfate reduction by plant cell suspension cultures

Cell suspension cultures of Catharanthus roseus (L.) G. Don were grown under S-auxotrophic (1.8 mM sulfate) and under S-heterotrophic (0.5 and 1.0 mM cysteine or methionine) conditions. The development of activity of the thiol sulfotransferases was followed during the complete growth period. Under auxotrophic growth, an NADPH-dependent S: sulfotransferase and a GSH-dependent S: sulfotransferase developed identically, whereas under heterotrophic growth, differences in the amount of enzymes and in the time course of their development occurred. The NADPH-dependent sulfotransferase appeared repressed by the S-amino acids but the GSH-dependent enzyme was derepressed. In that phenomenon, the development of the GSH sulfotransferase paralleled the development of the ATP-sulfurylase (EC 2774) activity of the cells.Abbreviations APS adenylylphosphosulfate - GSH reduced glutathione - PAPS phosphoadenylylphosphosulfate     

Biosynthesis of anthraquinones and related compounds in Galium mollugo cell suspension cultures

From Galium mollugo cell suspension cultures, 1,4-dihydroxy-3-prenyl-2-naphtholic acid methyl ester diglucoside was isolated along with anthraquinones and mollugin. Production of the diglucoside was much increased by administering 2-succinylbenzoate to the cultures. The incorporation of 2-succinylbenzoate into lucidin-3-primeveroside, mollugin and the diglucoside in the mode so far proposed for rubiaceous anthraquinones was verified by administration of ¹³C-labelled 2-succinylbenzoate to the cell cultures.     

Induction of benzoquinone formation by activated carbon in Lithospermum erythrorhizon cell suspension cultures

Cultured cells of Lithospermum erythrorhizon which were capable of producing red naphthoquinone (shikonin) derivatives on Linsmaier-Skoog's agar medium stopped synthesizing these compounds when grown in liquid medium without agar. However, when the liquid medium was supplemented with a small amount of activated carbon, the cells produced a new orange benzoquinone derivative, echinofuran B, which may be considered an abnormal metabolite of geranylquinol, the key intermediate in the biosynthesis of shikonin. A similar effect of activated carbon was also observed with a variant cell line incapable of producing shikonin derivatives even on the agar medium. By contrast, the callus cultures grown on the agar medium as well as the dried roots of the intact plant were found to contain a small amount of echinofuran C, another new benzoquinone related to echinofuran B, in addition to shikonin derivatives.     

An extracellular β-galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall-polysaccharide hydrolysis

β-Galactosidase (β-Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β-Galase (β-Galase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM-Sephadex C-50. DEAE-Sepharose CL-6B and Sephacryl S-200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S-200HR gel-permeation, and 60 kDa by SDS-PAGE after treatment with SDS and 2-mercaptoethanol. The pI was 6.5. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyranoside were 0.17 m M and 31.9 μmol (mg protein)^-1, h^-1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co²⁴, Cu²⁺, Hg^2-, p -chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo-fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.     

Stability enhancement of hGM-CSF in transgenic<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> suspension cell cultures

Proteolytic enzymes existing in plant cell cultured media are the major reason for the loss of secreted human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The addition of pepstatin, aprotinin and PMSF relatively decreased the proteolytic degradation of hGM-CSF in a conditioned medium, but sufficient prevention against the proteolytic activity could not be obtained with chemical protease inhibitors. Gelatin, as a competitive substrate for protease, showed a stabilizing effect in a conditioned medium. Compared to the initial hGM-CSF concentration in a conditioned medium, with 10 g/L of gelatin, 68% of the hGM-CSF remained after 5 days. In a cell culture experiment, 5 g/L of gelatin significantly stimulated the hGM-CSF production and accumulation in culture media, with no growth inhibition. Compared to the controls (4.72 μg/L), the extracellular hGM-CSF level could be increased to 39.78 μg/L with the addition of 5 g/L of gelatin.     

Transient expression of antibodies in suspension plant cell suspension cultures is enhanced when co‐transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing

Two distinct transient expression approaches were compared with assess the impact of the viral suppressor p19 on a recombinant protein production performed in Nicotiana benthamiana suspension culture. A parental N. benthamiana cell line was transiently transformed with either an Agrobacterium containing a gene construct for a murine IgG1 (R514) or concurrently with two Agrobacteria containing R514 or p19. In addition, a stably transformed N. benthamiana cell line that constitutively expresses p19 was transformed with R514‐containing Agrobacterium. The parental N. benthamiana cell line that had been co‐cultivated with both p19 and R514 achieved the highest yield of IgG1 (1.06 mg IgG1/kg FW; 0.024% TSP) compared with that obtained without p19 (0.61 mg IgG1/kg FW; 0.014% TSP). The N. benthamiana cell line that had been stably transformed with p19 only reached 0.25 mg IgG1/kg FW (0.009% TSP) when co‐cultured with R514‐containing Agrobacterium. Dual agroinfiltration of N. benthamiana leaves with p19 and R514 was also performed to assess for Agrobacteria efficiencies and 147.7 mg IgG1/kg FW were obtained. Therefore, our results demonstrate that transient co‐transformation of plant cell suspension culture with two transformation vectors is feasible and that the use of the viral suppressor of silencing p19 significantly raises the production of the protein of interest. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The accumulation of isocamptothecin A and B in suspension cell culture of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The initiation of the suspension culture of Camptotheca acuminata M42 cell line was reported. Isocamptothecin A (ICPTA) and isocamptothecin B (ICPTB), two alkaloids that do not exist in the parent plant were identified in the suspension cell culture of Camptotheca acuminata. Suspension cell culture was carried out in both flasks and a 5-l airlift bioreactor and the time course for cell growth and accumulations of ICPTA and ICPTB were investigated. It was found that accumulations of ICPTA and ICPTB was partly associated with cell growth and the size of cell aggregates affected the contents of ICPTA and ICPTB in the cultures to some extent (cell aggregates with a size of 2--4 mm in diameter presented the highest ICPTA and ICPTB contents). Although the cell dry weight of 15.1 g l^-1 achieved in a 5-l airlift bioreactor was lower than that in flasks, 18.4 g l^-1, the contents of ICPTA and ICPTB were almost the same levels.     

Increased hGM-CSF production and secretion with Pluronic F-68 in transgenic<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> suspension cell cultures

The effects of Pluronic F-68, a nonionic surfactant, on the production and secretion of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in a transgenicNicotiana tabacum cell suspension culture were investigated in this study. The addition of Pluronic F-68 was shown to extend cell survival in the stationary phase, but had no influence on effective initial cell growth. With regard to production, it increased the level of extracellular hGM-CSF two-fold. This may be attributable not only to the enhanced expression level, but also to the improved permeability of the cell membrane due to the interaction between Pluronic F-68 and the cell membrane and cell wall. The effect of Pluronic F-68 on the production and secretion of hGM-CSF in a bioreactor was also evaluated. hGM-CSF production in the bioreactor after the addition of Pluronic F-68 proved more effective than in flask cultures.     

Analysis of Messenger RNA Coding for S100 Protein in the Mammalian Brain

S100 protein is a brain-specific protein which is absent at birth and first appears in rabbit brain 2–3 days after birth. To determine how the synthesis of this brain-specific protein is regulated, mRNA was isolated from brain polysomes and assayed for S100 protein mRNA activity by in vitro translation in a heterologous cell-free system and immunoprecipitation of released polypeptides with rabbit anti-S I00 protein antiserum. 5100 protein mRNA was detected primarily in small polysomes containing five to eight ribosomes, and virtually no S 100 protein mRNA was present in polysomes containing more than eight ribosomes. S100 protein mRNA was not detected in brain polysomes at stages prior to the induction of synthesis of S100 protein, i.e., in fetal brain or in 1-day neonates. The amount of S100 protein mRNA in polysomes of the cerebral cortex and cerebellum was measured to see if it correlated with the level of S100 protein in the two regions of adult brain. The cerebellum, which contained three to four times the level of S100 protein in the cerebral cortex, contained four times more S100 protein mRNA.     

Interaction between abscisic acid and nitric oxide in PB90‐induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures

Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor‐induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up‐regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90‐induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor‐induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up‐regulation of Str and Tdc. ABA‐induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO‐induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90‐induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90‐induced catharanthine biosynthesis of C. roseus cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:994–1001, 2013     

Fused in Sarcoma (FUS) Protein Lacking Nuclear Localization Signal (NLS) and Major RNA Binding Motifs Triggers Proteinopathy and Severe Motor Phenotype in Transgenic Mice

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.