Ethephon- and Jasmonate-Elicited Pathogenesis-Related Ribonucleases in Cultured Ginseng Cells

Cultured ginseng cells (Panax ginseng C.A. Mey strain R-1) produce two proteins exhibiting RNase activity (Pg1 and Pg2), which, on the basis of their amino acid sequences, have been earlier referred to intracellular pathogenesis-related proteins. An immunoenzyme technique for estimation of these proteins was developed. A close correlation was found between the content of these proteins and the RNase activity of the cultured cells. Ethephon and jasmonic acid activated the RNase activity, ethephon being more efficient. Salicylic acid did not activate Pg1 and Pg2; high concentrations of salicylic acid suppressed the RNase activity of the culture. The protein kinase inhibitor, H-7, reduced the content and activity of RNases both in the presence and absence of ethephon. The results obtained permit a suggestion that ethylene and jasmonic acid signaling pathways, which include protein phosphorylation, are involved in the induction of PR-10 proteins.     

Ion Composition of Tobacco Cells Cultured under Sulfur Deficiency

In both photoheterotrophic and heterotrophic tobacco cells areduced supply of sulfate in the medium did not alter the ratebut the duration of exponential growth. The higher the sulfatesupply in the medium the longer exponential growth proceeded.However, the ion composition of photoheterotrophic and heterotrophiccells was affected by sulfur deficiency in completely differentways. The dynamics in the K⁺-, Na⁺-, Mg²*-, nitrate-, phosphate-,and malate-con-tents of photoheterotrophic cells during growthwere not at all, or only slightly changed, when the sulfatesupply in the medium was reduced from 1.8mM to 1.2 mM, 0.6 mM,or 0.3mM. In heterotrophic tobacco suspensions, however, severesulfur deficiency caused K⁺, Na⁺, Mg²⁺, and malate to accumulateand nitrate to begin to accumulate earlier inside the cells.Addition of sulfate after 4 days to heterotrophic suspensionsgrown under sulfur-limiting conditions prevented the accumulationof these cations and anions. During the initial period of growthalso phosphate accumulated inside heterotrophic tobacco cellsto amounts found to be the higher the smaller the sulfate-contentof the media. Apparently, in photoheterotrophic tobacco cellsthe ion composition can homeostatically be regulated independentfrom the cells' sulfate supply, whereas the ion compositionof heterotrophic tobacco cells appears to be highly dependenton the sulfate supply of the cells. ⁴Present address: Fraunhofer Institut für AtmosphärischeUmwéltforschung, Kreuzeckbahnstr. 19, D-8100 Garmisch-Partenkirchen, F.R.G. (Received August 30, 1988; Accepted January 18, 1989)     

Mineral composition of cultured Ginseng cells]

The contents of macroelements and microelements in ginseng roots and callus cultures was determined by atom absorption spectroscopy. Ginseng cells and tissues were shown to accumulate considerable amounts of microelements. The content of six of eleven mineral components studied (K, Ca, Na, Mo, Mn, and Cr) in callus cultures was higher than that in roots of agricultural ginseng plants. We revealed good correlations between the contents of microelements (K, Ca, and Mg), as well as between the concentrations of macroelements (Mo, Li, Cu, and Cr) in ginseng cultures. The ability to accumulate elements varied between ginseng species, which was probably related to their genetic features. Our findings indicate that cultured ginseng cells hold much promise as the source of microelements.     

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births^1,2, 60-80% of all miscarriages^3,4, 10% of stillbirths^2,5, 13% of individuals with congenital heart disease⁶, 3-6% of infertility cases², and in many patients with developmental delay and birth defects⁷. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance^8,9.  Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents^10-13.Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy''s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)^14,15.     

Mineral Composition of Human fascia lata

The mineral composition of pathologically unchanged human fascia lata was examined here using inductively coupled plasma optical emission spectrometry (ICP-OES) method for the first time. The total concentrations of Ag, Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, P, Pb, Sr, Ti, V and Zn were simultaneously measured in the tissue secured during autopsy. The age-related changes and between-gender differences in mineral composition of the examined tissue were investigated and discussed.     

Freeze Preservation of Cultured Plant Cells

A basic technique for successful freeze preservation and storage at -196°C of cultured plant cells and an assay of percentage survival following the freezing-storage-thawing procedure are described. These techniques have been applied to suspension cultures of carrot (3 cell lines), belladonna and sycamore. Dimethyl sulfoxide (DMSO) and glycerol, when appropriately applied, were the most effective cryoprotectants tested. Although these cryoprotectants were of low toxicity and did not cause alterations in the cytology and growth potential of the recovered cells, the cell lines differed in their sensitivity to the toxicity of these cryoprotectants. Small meristematic cells survived the freezing-thawing procedure better than larger more highly vacuolated cells. Specific differences in survival are in part explained in terms of differences in cell morphology.     

Somatic Embryogenesis in Cultured Carrot Cells

Somatic embryogenesis in cultured plant cells is an ideal system for investigating the whole process of differentiation and development from single cells to whole plants, and especially the molecular mechanism of expression of totipotency. This review reports recent progress the studies on somatic embryogenesis.     

Neurofilament Proteins in Cultured Chromaffin Cells

Antibodies were raised against the 200-kd, 145-kd, and 68-kd subunits of a rat neurofilament preparation. Immunoblots showed that each antibody was specific for its antigen and that it did not cross-react with any of the two other neurofilament polypeptides. Use of the three antibody preparations to stain bovine chromaffin cells in culture by the indirect immunofluorescence technique indicated that the three neurofilament polypeptides are present in chromaffin cells maintained in culture for 3 or 7 days. The three anti-neurofilament antibodies labelled the cells in a similar pattern: very thin filaments specifically localized around the nucleus were observed whereas neurites and growth cones, developed by cultured chromaffin cells, were generally not stained. Some fibroblasts were present in our cultures but they were never stained by any of the neurofilament antibodies. This indicated that the antibodies used do not react with vimentin, the major intermediate filament protein found in fibroblasts. The three neurofilament antibodies were also used to immunoprecipitate specifically three proteins of molecular weights 210 kd, 160 kd, 70 kd from solubilized extracts of cultured chromaffin cells that were radiolabelled with [35S]methionine. These proteins correspond in molecular weight to the neurofilament triplet found in bovine brain. Finally, the presence of neurofilaments in freshly isolated chromaffin cells was tested by immunoblotting using the 68-kd antibody. A 70-kd protein was specifically stained by this antibody, suggesting that neurofilaments are not only present in cultured chromaffin cells but also in the adrenal gland in vivo. It is concluded from these results that chromaffin cells contain completely assembled neurofilaments. This additional neuronal property again illustrates that chromaffin cells are closely related to neurons and therefore represent an attractive model system for the study of functional aspects of adrenergic neurons.     

Peroxisome Dynamics in Cultured Mammalian Cells

Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein–a mutated bacterial dehalogenase which is fused to the protein of interest–and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell-permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag-containing construct, at different wavelengths. Here, we show that peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of pre-existing peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half-life of approximately 2 days and are mainly degraded by an autophagy-related mechanism. The implications of these findings are discussed.     

Alcohol Dehydrogenase from Cultured Rice Cells

Rice cells in suspension culture had high alcohol dehydrogenaseactivity during the logarithmic growth phase (3rd to 5th day).Ethanol was accumulated both in the cells and in the medium.The highest amount of ethanol was accumulated on the 4th dayin cells (10 µmoles/g fresh weight) and during the stationarygrowth phase (8th day) (180 mM, ca. 1%) in the medium. The enzymewas isolated from the cell extract and purified 36-fold witha 14% yield by ammonium sulfate fractional precipitation, andchromatography on DEAE-Sephadex, Sephadex G-150 and Blue Dextran-Sepharose.The purified enzyme was homogeneous, as judged by its sedimentationvelocity, and poly acrylamide gel, starch gel and SDS-polyacrylamidegel electrophoreses. Its molecular weight was 76,000 distributedin two, identical 37,000 subunits. The isoelectric point wasat pH 5.5. The enzyme contained 2.1 g atoms of zinc, 12 freeSH groups and 3 to 4 SS bonds per molecule. The pH optimum forethanol oxidatioa was pH 9.5 and for acetaldehyde reductionpH 6.0. The K_m values for ethanol, NAD^$, acetaldehyde and NADHwere 64.5 mM, 47.1 µM, 1.3 mM and 9.5 µM. The aminoacid composition, substrate specificity, and the effects ofchelators, SH reagents and sugar metabolic intermediates alsoare reported. (Received August 25, 1981; Accepted December 7, 1981)     

Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.     

Membrane Potentials of Heterotrophically Cultured Tobacco Cells

The electrophysiological properties of the membrane of Nicotianatabacum var. Sarnsun cultured cells were determined using amicroelectrode technique in standard medium containing 1 mMKC1, 1 mM NaCl and 1 mu CaCl₂ at pH 7. Tobacco callus was derivedfrom the pith (E_m=–104.4%16.2 mV). The membrane potentialsof the callus cells did not show a symmetrical Gaussian distributionbut were scattered over a wide range. The percentage of highmembrane potential cells increased as the subculture was continueduntil about 11 months and then decreased. The response of themembrane potential to electric stimulus, ionic composition,metabolic inhibitors, sugars and amino acids was characteristicof high (E_m=–{small tilde}–160 220 mV; H-cells)and low (E_m=–80{small tilde}–90 mV; L-cells) membranepotential cells. The membrane potential of H-cells was largelydepolarized by addition of CN^–, carbonium cyanide m-chlorophenylhydrazone,decyclohexylcarbodiimide, and triphenyltin chloride and transientlydepolarized by addition of glucose, galactose, mannose or sucrose,and D-alanine, L-alanine or Llysine, but the membrane potentialof L-cells was not. (Received December 3, 1982; Accepted March 16, 1983)     

Glucosylation of Vanillin by Cultured Plant Cells

Vanillin was converted into the corresponding glucoside in suspension-cultured cells of Ceffea arabica. The maximum efficiency of glucosylation was 85% within 24 h after the addition of 1 mM vanillin when cultured in a modified Murashige and Skoog’s medium with 5 µM 2,4-dichlorophenoxyacetic acid and 0.5 µM kinetin. The glucoside was identified as 4-formyl-2-methoxyphenyl-O-β-D-glucopyranoside by ¹H-NMR, ¹³C-NMR, FAB-MS, and hydrolysis by α- and β-glucosidases. It retained the antimutagenic and antimicrobial activities of vanillin.     

The Dynamics of Microtubules in Cultured Cells

The behavior of microtubules in cultured cells in a cooled matrix after the microinjection of fluorescent tubulin was studied using a frame recording with a digital camcorder. In the cell lamella, the positive ends of individual microtubules extend and shorten at random. The histograms of rate distribution have an almost normal distribution with a mode close to 0. The maximum rate of lengthening and shortening reaches 30 and 50 m/min, respectively. The positive ends of microtubules in PtK cells were in an equilibrium state, while in murine embryonic fibroblasts and Vero cells, they were usually displaced to the cell edge. Free microtubules were present in the cells of all three cultures. In the epithelial cells, they were numerous and relatively stable, while in the fibroblasts, they occurred rarely and were depolymerized at the proximal end. Free microtubules in PtK cells appeared mostly due to spontaneous assembly in the cytoplasm (not in the relationship with the preexisting microtubules) and, more rarely, due to breakage of long microtubules. Separation of microtubules from the centrosome is a very rare event. Unlike positive ends that were characterized by dynamic instability, negative ends were stable and were sometimes depolymerized. When long microtubules were broken, new negative ends were formed that were, as a rule, stable, while in the lamella of fibroblasts (in murine embryonic fibroblasts and Vero cells), new negative ends were immediately depolymerized: free microtubules existed in these cells no more than 1–2 min. A diffusion model has been proposed where the behavior of microtubule ends is considered as unidimensional diffusion. The coefficient of diffusion of positive ends in the epithelial cells is several times less than in the fibroblasts, thus suggesting a higher rate of tubulin metabolism in the fibroblasts as compared to the epithelium. The results obtained indicate that for the exchange of long microtubules, the dynamic instability is not sufficient. In the fibroblasts, their exchange takes place mostly at the expense of depolymerization of the liberating negative ends, which agrees with the previously proposed conveyer hypothesis of microtubule assembly on the centrosome.     

Post-Translational Arginylation of Proteins in Cultured Cells

The aim of this study was to analyze the N-terminal post-translational incorporation of arginine into cytosolic proteins from cultured cells and the in vitro incorporation of arginine into soluble proteins of PC12 cells after serum deprivation. Arginine incorporation was measured in the presence of protein synthesis inhibitors. None of the inhibitors used affected significantly the arginylation reaction while the novo synthesis of protein was reduced by 98%. Under these conditions, we found that of the total [¹⁴C]arginine incorporated into the proteins, around 20% to 40% was incorporated into the N-terminal position of soluble proteins by a post-translational mechanism. These results suggest that this post-translational aminoacylation may be a widespread reaction in neuronal and non-neuronal cells. We also found that in PC12 cells, the in vitro post-translational arginylation was 60% higher in apoptotic cells with respect to control cells. These findings suggest that the post-translational arginylation of proteins may be involved in programmed cell death.     

Surface Proteins of Cultured Mouse Cerebellar Cells

Surface proteins of cultured monolayer cells from embryonic and early postnatal C57BL/6J mouse cerebella were identified by a lactoperoxidase-catalysed ¹³¹iodine labelling technique. Major iodinated polypeptides have molecular weights of approximately 200, 145, 120, 100, 85, 65, 50, and 30 X 10³ (P200, P145, ?) as estimated by sodium dodecylsulphate polyacrylamide gel electrophoresis. Membrane glycoproteins, of apparent molecular weights 200, 145, 100, 85, and 50 X 10³, are detected by biosynthetic labelling with [³H]fucose. The two major iodinated proteins are the glycoproteins P200 and P145. P145 is released from the cells into the medium together with other surface proteins. No changes in the patterns of labelled cerebellar cell surface proteins are detectable between embryonic day 17 and postnatal day 10. A pattern similar to the one seen with cerebellum is obtained with embryonic day 12 and 17 cerebral cortex. Cultured retinal cells from 2-day-old mice, skin fibroblasts, and l -cells display a distinctly different pattern, which does not contain P145 as a major iodinated component. In granule cell-enriched fractions of cerebellar cells the two glycoproteins P200 and P145 are proportionately increased, while three proteins, P100, P85, and P50, are more abundant in the glial cell-enriched fraction. These three polypeptides are also enriched in cells obtained from staggerer mutant mice. An antiserum against 4-day-old cerebellar cells (anti-NS-4) precipitates the 145 and 200 X 10³ molecular weight proteins, from lysates of both embryonic cerebral and postnatal cerebellar cells. From lysates of mouse retinal cells, anti-NS-4 antiserum precipitates two proteins with molecular weights of 140 and 210 X 10³. Rohrer H. and Schachner M. Surface proteins of cultured mouse cerebellar cells. J. Neurochem. 35, 792–803 (1980).     

Ultrastructure of Cells Cultured on Polycarbonate Membranes

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 pm or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Ginseng Berry Extract Promotes Maturation of Mouse Dendritic Cells

Ginseng extract has been shown to possess certain anti-virus, anti-tumor and immune-activating effects. However, the immunostimulatory effect of ginseng berry extract (GB) has been less well characterized. In this study, we investigated the effect of GB on the activation of mouse dendritic cells (DCs) in vitro and in vivo. GB treatment induced up-regulation of co-stimulatory molecules in bone marrow-derived DCs (BMDCs). Interestingly, GB induced a higher degree of co-stimulatory molecule up-regulation than ginseng root extract (GR) at the same concentrations. Moreover, in vivo administration of GB promoted up-regulation of CD86, MHC class I and MHC class II and production of IL-6, IL-12 and TNF-α in spleen DCs. GB also promoted the generation of Th1 and Tc1 cells. Furthermore, Toll like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling pathway were essential for DC activation induced by GB. In addition, GB strongly prompted the proliferation of ovalbumin (OVA)-specific CD4 and CD8 T cells. Finally, GB induced DC activation in tumor-bearing mice and the combination of OVA and GB treatment inhibited B16-OVA tumor cell growth in C57BL/6 mice. These results demonstrate that GB is a novel tumor therapeutic vaccine adjuvant by promoting DC and T cell activation.     

Digitoxin Biosynthesis in Isolated Mesophyll Cells and Cultured Cells of Digitalis

In the laminae of Digitalis, most of the digitoxin present isfound in the mesophyll. A new method for determining the amountof digitoxin biosynthesis using a digitoxin antibody was devisedto estimate this activity in isolated mesophyll cells and culturedcells. Isolated mesophyll cells showed significant activity,which suggests that the site of biosynthesis and the accumulationof cardenolides in a lamina of Digitalis is mainly in the mesophyllcells. Of five liquid cultures of D. purpurea; green shoot-formingcultures, white shoot-forming cultures, root-forming cultures,undifferentiated green cells and undifferentiated white cells,the green shoot-forming cultures had the highest activity. Thewhite shoot-forming cultures had about one-third the activityof the green shoot-forming cultures, and the other three cultureshad very low activity. No stimulatory effect of light was foundduring the 48-h incubation. (Received January 19, 1984; Accepted June 8, 1984)     

Tissue Cultured Mountain Ginseng Adventitious Roots™: Safety and Toxicity Evaluation

Panax ginseng has strong anticancer, antidiabetic, antioxidant activity, various physiological functions, and is used as a nutritional and medicinal supplement. Tissue cultured mountain ginseng adventitious roots? (TCMGARs) powder, which is derived from commercial scale bioreactor cultures, was tested for its consumption safety. The reverse mutation, chromosomal aberration and the micronucleus tests were found to show no significant mutagenicity. Furthermore, thirteen weeks of repeated dose toxicity of TCMGARs oral doses from 300–900 mg/kg, with a four‐week recovery period, did not produce mortality or significant changes in the general behavior and gross appearance of the internal organs of rats. The absolute body weight, urine test, haematology, blood chemistry, absolute organ weight and histopathological examination of both main and recovery groups revealed that there were no differences between the control and the treated rats. These results confirm that TCMGARs are safe and nontoxic at an average dietary consumption level.