Steroidal Saponins are not Main Building Units of the Prolamellar Body in Etioplasts

Sugar-containing lipids were analyzed by thin layer chromatographyin various cell fractions of etiolated Aoena leaves, Auena intactetioplasts and prolamellar bodies isolated from Cucurbita etioplasts.We confirmed the presence of steroidal saponins, avenacosidesA and B in etiolated leaves and crude etioplast fraction ofAvena, but scarcely detected them in Avena intact etioplastspurified by Percoll density gradient centrifugation. Saponinswere hardly detected in the paracrystalline prolamellar bodiesfrom Cucurbita etioplasts. We concluded that steroidal saponinsare not main building units of the prolamellar body in the etioplasts. (Received August 5, 1982; Accepted February 15, 1983)     

Red light induced production of gibberellin-like substances in homogenates of etiolated wheat leaves and in suspensions of intact etioplasts

Summary Homogenates of etiolated wheat leaves contain increased levels of acidic gibberellin (GA)-like substances following treatment with red light. Differential centrifugation of homogenates indicates that the response is confined to the 1000 g (or plastid) fraction. Irradiation of suspensions of intact etioplasts also increases the level of extractable GA-like activity. Phytochrome can be detected spectrophotometrically in preparations of etioplasts. The response in etioplasts can be inhibited by chloramphenicol, but not by cycloheximide, and partially by Amo 1618. The GA-like substances produced in etioplasts seem capable of passing into the surrounding medium within 20 min.Abbreviation GA Gibberellin     

Phytochrome controlled gibberellin metabolism in etioplast envelopes

Summary Disruption of preparations of intact etioplasts isolated from wheat (Triticum aestivum L.) leaves in the dark leads to an increase in the level of extractable gibberellin-like (GA-like) activity. Irradiation of disrupted preparations with red light causes an alteration in the Rf of part of the GA-like activity. A study of sub-etioplast fractions prepared by gradient centrifugation indicates that the phytochrome and the GA-like substances are associated principally with the etioplast envelope.Abbreviations GA gibberellin - MOPS N-morpholino-3-propanesulphonic acid - EDTA ethylene diamine tetra-acetic acid - ATP adenosine triphosphate - R red light     

In vitro primary allo-CTL generation by enucleated antigen-presenting cells

It is generally thought that only viable cells can elicit a primary cytotoxic T-lymphocyte (CTL) response. We present evidence that this is not so, since enucleated tumor cells can generate a strong cytolytic response of unprimed allogeneic human T lymphocytes. Cytoplasts (enucleated cells) were obtained by incubation with cytochalasin B and subsequent isopycnic centrifugation. Their purity was assessed by electron microscopy and flow cytometry. Membrane fractions were prepared by nitrogen cavitation, and used in parallel with cytoplasts and intact cells as stimulators in primary allo-CTL generation; although all cell fractions expressed high amounts of class I and II histocompatibility antigens, as assessed by flow cytometry and ELISA technique, only the cytoplasts generated a strong cytotoxic response of naive peripheral T cells, like that induced by intact cells. The dogma that an intact and metabolically active stimulator cell is required for the primary generation of CTLs is questioned.     

Etioplast Development in Dark-grown Leaves of Zea mays L

The ultrastructure of etioplasts and the acyl lipid and the fatty acid composition of sequential 2-centimeter sections cut from the base (youngest) to the top (oldest) of nonilluminated 5-day-old etiolated leaves of Zea mays L., and the acyl lipid and fatty acid composition of the etioplasts isolated from them have been investigated. There is a 2.5-fold increase in the size of the plastids from the base to the tip of the leaf, and an increase both in the size of the prolamellar body and in the length of lamellae attached to it. The etioplasts in the bundle sheath and mesophyll cells of the older, but not the younger leaf tissue, are morphologically distinct. The monogalactosyl and digalactosyldiglycerides, phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol were the only detectable acyl lipids in the isolated etioplast fractions. Together with phosphatidylethanolamine these were also the major acyl lipids in the whole leaf sections. With increasing age of the leaf tissue, increases occurred in two of the major plastid lipids, monogalactosyldiglyceride and phosphatidylglycerol, while the levels of essentially nonplastid lipids remained constant or declined slightly. The monogalactosyldiglyceride to digalactosyldiglyceride ratio increased from 0.4 to 1.1 in the tissue sections of increasing age and from 0.7 to 1.2 in the etioplasts isolated from them. Similarly, the galactolipid to phospholipid ratio increased from 0.8 to 1.4 in the tissue and from 0.5 to 4.5 in the isolated plastids. In the latter, the proportions of phosphatidylglycerol (as a per cent of total phospholipid) increased from 20 to 41% with increasing age of plastids.

Linolenic acid was the major fatty acid in the total lipid of each of the etioplast fractions, but it was only the major fatty acid in the total lipid of the oldest leaf tissue. Its proportion in both total lipid extracts and individual lipids increased with age. The trans Δ³ hexadecenoic acid was absent from all lipids. The protochlorophyllide content of the tissue increased with age. The results are discussed in relation to the use of illuminated etiolated leaves for studying chloroplast development.

     

Physical separation of hemopoietic stem cells from cells forming colonies in culture

Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.     

Isolation of anucleate cells using a fluorescence activated cell sorter (FACS II)

Human fibroblasts or mouse teratocarcinoma cells were enucleated by density gradient centrifugation in the presence of cytochalasin B (CB). The resulting mixed population of nucleated and anucleate cells was further purified by flow sorting, using the dye Hoechst 33342 as a fluorescent label for the nucleated cells. The purity of the anucleate cells obtained with this technique was at least 99%, as was shown by histological staining of the sorted fractions. Sorted enucleated fibroblasts were shown to have an intact cell membrane as indicated by their ability to convert fluorescein diacetate into fluorescein and to accumulate this product. They were found to attach and spread when cultured and showed protein synthesis immediately after enucleation, evidenced by the incorporation of [³H]leucine. Sorted enucleated teratocarcinoma cells also had an intact cell membrane, but they did not attach when cultured.     

Fatty acid biosynthesis in mitochondria of grasses: malonyl-coenzyme A is generated by a mitochondrial-localized acetyl-coenzyme A carboxylase

We present biochemical evidence for the occurrence of a 250-kD multifunctional acetyl-coenzyme A carboxylase in barley (Hordeum vulgare) mitochondria. Organelles from 6-d-old barley seedlings were purified by differential centrifugation and Percoll density gradient centrifugation. Upon analysis by two-dimensional Blue-native (BN)/SDS-PAGE, an abundant 250-kD protein can be visualized, which runs at 500 kD on the native gel dimension. A similar 500-kD complex is present in etioplasts from barley. The mitochondrial 250-kD protein is biotinylated as indicated by specific reaction with an antibody directed against biotin. Peptide sequence analysis by electrospray ionization tandem mass spectrometry of the 250-kD proteins from both organellar fractions revealed amino acid sequences that are 100% identical to plastidic acetyl-coenzyme A carboxylase from wheat (Triticum aestivum). The 500-kD complex was also detected in wheat mitochondria, but is absent in mitochondrial fractions from Arabidopsis. Specific acetyl-coenzyme A carboxylation activity in barley mitochondria is higher than in etioplasts, suggesting an important role of mitochondria in fatty acid biosynthesis. Functional implications are discussed.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Structure and function of developing barley plastids

Five different regions of the first foliage leaf of etiolated barley seedlings were studied with respect to leaf growth, plastid growth and replication, differentiation of etioplasts, and conversion of etioplasts into chloroplasts upon illumination. Ultrastructural changes of the plastids were correlated with chlorophyll synthesis and development of photosynthetic activity as measured by (14)CO(2) incorporation and O(2) evolution. The first foliage leaf has greater linear growth over a longer period of time in the dark than in the light. Only the bottom two regions (4 and 5) are still growing in the 5-day etiolated leaf. Region 4 grows by cell elongation, and region 5 grows by both cell division and elongation. Plastids in all five regions of the leaf are capable of enlarging when exposed to light. This is true both for the intact plant and for excised sections. Plastid replication occurs predominantly in the younger regions of the leaf (regions 3, 4, and 5). The amount of chlorophyll synthesized by different regions in the intact plant is significantly higher (3-40 times) than that made by excised sections. Ultrastructural changes occurring in each region when excised sections are illuminated were classified into five stages involving increased membrane synthesis and appression into grana, and these changes were correlated with the first appearance of photosynthetic activity. The earliest detectable photosynthetic activity occurs in region 1 after 2 hours of illumination when chloroplasts show only a few overlaps in the thylakoids. Plastids in younger regions of the leaf require up to 24 hours of light to form grana and develop photosynthetic activity. Plastids in each region of the leaf are in different stages of development when photosynthesis is initiated, indicating that development of photosynthetic activity is not strictly correlated with a certain stage of plastid development. Membrane appression is not indicative of photosynthetic activity since overlaps are formed in the dark, but it was always present when photosynthetic activity was detectable. Likewise, there does not appear to be any strict correlation between the presence of chlorophyll and membrane appression. These results show that the particular structural and functional correlations that can be made depend to a large degree on age of the tissue.     

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.     

Changes in distribution of nucleoids in developing and dividing chloroplasts and etioplasts ofArena sativa

Summary Nucleoid distribution in chloroplasts and etioplasts at the different developmental stages was examined with the first leaves ofAvena sativa by using a DNA-specific fluorescent probe, 46-diamidino-2-phenylindole (DAPI). In light-grown first leaves, three types of plastid nucleoid distribution were recognized. 1. Peripheral distribution in undeveloped chloroplasts which contain only a few thylakoids in the middle region of the leaf sheath. 2. Ring-like arrangement along the rim of developing and dividing young chloroplasts, of which grana were composed of four to eight layers of thylakoids, at the base of the leaf blade. The plane of the nucleoids' ring is in parallel with the face of the thylakoids. 3. Scattered distribution of 10 to 20 discrete spherular nucleoids in the stroma of fully developed chloroplasts, of which grana were composed of up to 20 thylakoids, in the regions of the middle and the tip of the leaf blade. In dark-grown first leaves two types were recognized. 1. Peripheral distribution in developing and dividing young etioplasts in the leaf sheath and the base of the leaf blade. 2. Scattered distribution of 10 or more discrete spherular nucleoids in fully developed etioplasts, containing extended prothylakoids, in the regions of the middle and the tip of the leaf blade. Ring-like arrangement of nucleoids was not observed in any etioplasts. The results indicates that spatial arrangement of plastid nucleoids dynamically changes in close relationship with the development of the inner membrane systems of plastids.     

Subcellular localization of chorismate-mutase isoenzymes in protoplasts from mesophyll and suspension-cultured cells of Nicotiana silvestris

The subcellular locations of two readily discriminated chorismate-mutase (EC 5.4.99.5) isoenzymes from Nicotiana silvestris Speg. et Comes were determined in protoplasts prepared from both leaf tissue and isogenic suspension-cultured cells. Differential centrifugation was used to obtain fractions containing plastids, a mixture of mitochondria and microbodies, and soluble cytosolic proteins. Isoenzyme CM-1 is sensitive to feedback inhibition by l-tyrosine and comprises the major fraction of total chorismate mutase in suspension-cultured cells. Isoenzyme CM-2 is not inhibited by l-tyrosine and its expression is maximal in organismal (leaf) tissue. Isoenzyme CM-1 is located in the plastid compartment since (i) proplastids contained more CM-1 activity than chloroplasts, (ii) both chloroplast and proplastid fractions possessed the tyrosine-sensitive isoenzyme, and (iii) latency determinations on washed chloroplast preparations confirmed the internal location of a tyrosine-sensitive isoenzyme. Isoenzyme CM-2 is located in the cytosol since (i) the supernatant fractions were heavily enriched for the tyrosineinsensitive activity, and (ii) a relatively greater amount of tyrosine-insensitive enzyme was present in the supernatant fraction derived from organismal tissue.     

Activity of Membrane-Associated Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase during Development of the Inner Membrane System in Potato Chloroplasts

Membranes of etioplasts and chloroplasts from potato plants (Solanum tuberosumL., cv. Zhu-kovskii) were fractionated by centrifugation in a sucrose gradient, and the fractions of primary membranes, fretlike structures, and frets and granal thylakoids were isolated. The carboxylase and oxygenase activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were measured in these fractions. The membrane-bound Rubisco (mRubisco) was reconstructed in the artificial system from the primary membranes and the soluble form of Rubisco (sRubisco), both isolated from the chloroplasts. The higher carboxylase and lower oxygenase activities were found to be characteristic of this reconstructed mRubisco as compared to sRubisco. The degree of sRubisco association with primary membranes depended on the Mg²⁺concentration. The data suggested that the association of sRubisco with primary membranes occurred at the early stages of membrane formation. The biological role of mRubisco is assumed to consist in controlling the ratio between photosynthesis and photorespiration at the membrane level in plants of various physiological states.     

Role of Silicon in Diatom Metabolism

The in vivo uptake of ³¹Si-silicic acid and ⁶⁸Ge-germanic acid by cell organelles of Nitzschia alba Lewin and Lewin and Cylindrotheca fusiformis Reimann and Lewin was demonstrated. The organelles were isolated by fractionation of pre-labeled cells by differential centrifugation in 0.4 M sucrose medium. Electron micrographs showed that the isolated organelles were intact, with the exception of the N. alba mitochondria which appeared swollen and the C. fusiformis chloroplasts which had ruptured outer membranes and lacked stroma material. The amount of ³¹Si or ⁶⁸Ge per mg protein of the subcellular fractions decreased in the following general order for both organisms: cell wall, mitochondria, chloroplasts, vesicles, and microsomes. A portion of the ³¹Si or ⁶⁸Ge in the organelle fractions could be extracted into a distilled water wash. The uptake of silicon by the organelles suggests that silicon may be involved in some processes of the compartmentalized systems of the cell.     

Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar ‘Villafranca’) cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml^?1, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml^?1 and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin‐induced NO production was sensitive to sodium azide and N^G‐monomethyl‐l ‐arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells.     

Steroidal saponins in etiolated,greening and green leaves and in isolated etioplasts and chloroplasts ofAvena sativa

Summary New results obtained in course of studies on steroidal saponins of oat indicate that in contrast to our former conclusions the prolamellar body (PLB) is not built up by steroidal saponins. Saponin content of whole leaves is similar in green as well as in etiolated leaves and does not change significantly during development of leaves. In contrast to chloroplast, isolated purified etioplasts contain high amounts of the desgluco-avenacosides, which were thought to be responsible for the tubular structure of PLBs. The amount of these PLB-saponins, however, is 10–60 times higher in isolated etioplasts than in etiolated leaves. Evidence is presented that the PLB-saponins originate from leaf-saponins during disruption of cells and attach to etioplasts or PLBs. This attachment to PLB-components explains our former results that the PLB-destruction during greening is accompanied by a decline in the amount of the desgluco-avenacosides in isolated PLB-fractions of greening leaves.     

Proteome analysis of the rice etioplast: metabolic and regulatory networks and novel protein functions

We report an extensive proteome analysis of rice etioplasts, which were highly purified from dark-grown leaves by a novel protocol using Nycodenz density gradient centrifugation. Comparative protein profiling of different cell compartments from leaf tissue demonstrated the purity of the etioplast preparation by the absence of diagnostic marker proteins of other cell compartments. Systematic analysis of the etioplast proteome identified 240 unique proteins that provide new insights into heterotrophic plant metabolism and control of gene expression. They include several new proteins that were not previously known to localize to plastids. The etioplast proteins were compared with proteomes from Arabidopsis chloroplasts and plastid from tobacco Bright Yellow 2 cells. Together with computational structure analyses of proteins without functional annotations, this comparative proteome analysis revealed novel etioplast-specific proteins. These include components of the plastid gene expression machinery such as two RNA helicases, an RNase II-like hydrolytic exonuclease, and a site 2 protease-like metalloprotease all of which were not known previously to localize to the plastid and are indicative for so far unknown regulatory mechanisms of plastid gene expression. All etioplast protein identifications and related data were integrated into a data base that is freely available upon request.     

Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts

Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues.